Anticoagulant Pathway Research Articles

Protein C System Activity after Physical Exercise: Possible Thrombophilic Implications

The role of influence on protein C anticoagulant system and on PC deficiency-related trombophilic risk due to strenuous physical exercise is still under discussion. In order to investigate the modification of the protein C anticoagulant pathway after strenuous exercise, we measured ProC® Global assay, a protein C activity dependent clotting time, in 10 healthy subjects before and immediately after maximal treadmill exercise, and at 5, 15, 30 and 60 min in the recovery phase. The most evident change was a shortening of ProC ®Global clotting time from a basal values of 123 sec to 84 sec at 30 min in post exercise. Our study shows that the coagulation unbalance observed after strenuous exercise and with no consequence in healthy individuals with normal PC level, could increase the thrombophilic risk in silent carriers of major defects of the protein C system and occasionally trigger an episode of deep vein thrombosis.

The Immunoregulatory Activities of Activated Protein C in Inflammatory Disease.

Tissue injury prompts the initiation of host defense responses to limit blood loss, restrict pathogen entry, and promote repair. Biochemical and cellular pathways that lead to blood coagulation serve a fundamental role in generating a physical barrier at the wound site, but have also evolved to promote immune response to injury. Similarly, anticoagulant pathways that attenuate clot formation also regulate innate and adaptive immune responses. Of particular importance is activated protein C (APC) which serves as a principal regulator of thrombin generation, shapes the innate immune response to infection, and has been shown to contribute to the adaptive immune response in several preclinical models of autoimmune disease. APC controls blood coagulation by proteolytic degradation of procoagulant activated cofactors essential for fibrin clot development, but also cleaves multiple additional substrates and interacts with cell surface receptors to exert additional physiologically important roles. In this review, we focus on the molecular mechanisms utilized by APC to limit inflammation and, in particular, current understanding of the basis for APC anticoagulant and signaling activities. In particular, we provide an overview of established and emerging signaling pathways initiated by APC on endothelial cells, monocytes, neutrophils, dendritic cells, and T cells to control and regulate immune cell physiology. Finally, we consider the impact of APC activity in the context of both acute and chronic inflammatory disease, and the continuing efforts to harness the immunoregulatory properties of recombinant APC for therapeutic use.

Recombinant and chemo-/bio-orthogonal synthesis of liposomal thrombomodulin and its antithrombotic activity

Thrombomodulin (TM) is an endothelial cell membrane protein that acts as a major cofactor in the protein C anticoagulant pathway. The EGF-like domains 4-6 of TM (TM456) are essential for PC activation. In this study, we proposed a liposomal recombinant TM conjugate to mimic the membrane TM structure and its anticoagulant activity. First, a DSPE-PEG2000-TM456 was successfully synthesized by site-specific conjugation of azido-TM456 with DSPE-PEG2000-DBCO via copper-free click chemistry quantitatively. Then, liposome-TM456 was fabricated via direct liposome formation with the DSPE-PEG2000-TM456 and other lipids. This liposomal formulation of TM456 retained protein C activation activity as that of TM456. Also, liposome-TM456 was much more stable and had a longer plasma half-life than TM456 and DSPE-PEG2000-TM456, respectively. Moreover, liposome-TM456 showed invivo anticoagulant effect by decreasing the mortality from 80% to 20% in a thrombin-induced thromboembolism mouse model. The reported liposome-TM456 conjugate mimics the endothelial TM anticoagulation activity and may serve as an effective anticoagulant agent candidate for future development.

Novel insights into the regulation of coagulation by factor V isoforms, tissue factor pathway inhibitorα, and protein S

Factor V (FV) is a regulator of both pro- and anticoagulant pathways. It circulates as a single-chain procofactor, which is activated by thrombin or FXa to FVa that serves as cofactor for FXa in prothrombin activation. The cofactor function of FVa is regulated by activated protein C (APC) and protein S. FV can also function as an anticoagulant APC cofactor in the inhibition of FVIIIa in the membrane-bound tenase complex (FIXa/FVIIIa). In recent years, it has become clear that FV also functions in multiple ways in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. Of particular importance is a FV splice variant (FV-Short) that serves as a carrier and cofactor to TFPIα in the inhibition of FXa. FV-Short is generated through alternative splicing of exon 13 that encodes the large activation B domain. A highly negatively charged binding site for TFPIα is exposed in the C-terminus of the FV-Short B domain, which binds the positively charged C-terminus of TFPIα, thus keeping TFPIα in circulation. The binding of TFPIα to FV-Short is also instrumental in localizing the inhibitor to the surface of negatively charged phospholipids, where TFPIα inhibits FXa in process that is stimulated by protein S. Plasma FV activation intermediates and partially proteolyzed platelet FV similarly bind TFPIα with high affinity and regulate formation of prothrombinase. The novel insights gained into the interaction between FV isoforms, TFPIα, and protein S have opened a new avenue for research about the mechanisms of coagulation regulation and also for future development of therapeutics aimed at modulating coagulation.

Factor VIIa interaction with EPCR modulates the hemostatic effect of rFVIIa in hemophilia therapy: Mode of its action.

Recent studies established that clotting factor VIIa (FVIIa) binds endothelial cell protein C receptor (EPCR). It has been speculated that FVIIa interaction with EPCR might augment the hemostatic effect of rFVIIa in therapeutic conditions. The present study is carried out to investigate the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of rFVIIa in hemophilia therapy. Active-site inhibited FVIIa, which is capable of binding to EPCR but has no ability to activate factor X, reduced the concentration of rFVIIa required to correct the bleeding following the saphenous vein injury in mouse hemophilia model systems. Higher doses of rFVIIa were required to restore hemostasis in EPCR overexpressing hemophilia mice compared to hemophilia mice expressing normal levels of EPCR. Administration of FVIII antibody induced only mild hemophilic bleeding in EPCR-deficient mice, which was corrected completely with a low dose of rFVIIa. Administration of therapeutic concentrations of rFVIIa increased plasma protein C levels in EPCR overexpressing mice, indicating the displacement of protein C from EPCR by rFVIIa. EPCR levels did not significantly alter the bioavailability of rFVIIa in plasma. Overall, our data indicate that EPCR levels influence the hemostatic effect of rFVIIa in treating hemophilia. Our present findings suggest that FVIIa displacement of anticoagulant protein C from EPCR that results in down-regulation of activated protein C generation and not the direct effect of EPCR-FVIIa on FX activation is the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of rFVIIa in hemophilia therapy.

Abstract 142: Biomimetic Recombinant Thrombomodulin Conjugate and Its Anticoagulant Activity

Thrombomodulin (TM) is an endothelial cell membrane protein that acts as a major cofactor in the protein C (PC) anticoagulant pathway. To closely mimic membrane protein structural feature of TM, we proposed a membrane-mimetic re-expression of recombinant TM onto liposome. The EGF-like domains 4-6 of TM (TM 456 ) are essential for PC activation. A recombinant TM containing TM 456 and an azidohomoalanine at C-terminus was expressed in E. coli . The biomimetic liposomal recombinant TM conjugate was prepared by conjugation of the recombinant TM 456 -azide with liposome via orthogonal chemistry and confirmed with Western blotting and PC activation activity. The liposomal recombinant TM 456 conjugate showed a 2-fold higher PC activation activity than that of the recombinant TM 456 alone , which indicated that the lipid membrane has a beneficiary effect on the recombinant TM 456 ’s activity. Also, the liposomal recombinant TM 456 conjugate showed higher stability and longer plasma half-life than TM 456 . Moreover, the liposomal recombinant TM 456 conjugate showed in vivo anticoagulant activity by decreasing the mortality in a thrombin-induced thromboembolism mouse model. The reported liposomal TM 456 conjugate mimics endothelial TM structure and anticoagulant activity and may serve as an anticoagulant agent candidate for future development.

Genetic factors in pediatric venous thromboembolism

Venous thromboembolism (VTE) is the predominant form of thrombosis in children and exhibits a strong genetic background as observed by familial aggregation. While data from diverse genome wide association studies (GWAS) and meta-analyses based on large study samples exist for adult patients, most reports on VTE in children rely on candidate gene studies based on small family collectives. However, results from adult patients cannot be directly transferred to children due to differences in their coagulation system and applicable environmental factors. By now, genetic variants in 8 genes have been described in the context of pediatric VTE, most of them connected to anticoagulation pathways. Since all these variants taken together cannot fully explain the observed heritability in pediatric VTE, further studies in pediatric study collectives are needed. Recently, the first GWAS on pediatric VTE has been carried out pointing towards vesicle processing in blood cells as process involved in pediatric VTE.

Evaluation of the anticoagulant potential of polysaccharide-rich fractions extracted from macroalgae

The aim of this study was to evaluate the potential anticoagulant activity of sulphated polysaccharide-containing extracts of six french edible marine macroalgae. Aqueous extracts of brown (Himanthalia elongata, Laminaria digitata, Ascophyllum nodosum, Fucus vesiculosus), green (Ulva lactuca) and red (Chondrus crispus) macroalgae were prepared and their biochemical properties were determined, including major biomolecules, sulphate and ash contents. The anticoagulant activity of each extract was investigated using different scales from the specific antithrombin-dependent pathway (anti-Xa and anti-IIa) to the intrinsic and/or common (Activated Partial Thromboplastin Time, APTT), extrinsic (Prothrombin Time, PT) or common (Thrombin Time, TT) anticoagulant pathways, and compared with those of commercial anticoagulants, heparin and Lovenox®. Laminaria digitata, Fucus vesiculosus and Chondrus crispus extracts showed a significant APTT anticoagulant capacity, only 5-fold lower than that of Lovenox®, which is a pure low molecular weight heparin used as an anticoagulant agent to prevent pulmonary embolism in patients undergoing surgery.

The Rebalanced Hemostasis System in End-stage Liver Disease and Its Impact on Liver Transplantation.

There are 3 physiological phases of action in the modern view of hemostasis. Primary hemostasis begins with endothelial injury, exposure of von Willebrand factor (vWF), platelet activation and aggregation, and an initial thrombin “burst.” Coagulation then follows with activation of the coagulation cascade, through extrinsic and intrinsic pathways in a positive feedback loop activated by thrombin and the formation of the fibrin mesh and fortification of the classic clot. Lastly, fibrinolysis must occur through the action of anticoagulant proteins and enzymes to avoid pathologic thrombus formation. Primary hemostasis is typically measured through platelet counts, vWF levels, platelet function assays, and bleeding time. Coagulation is generally measured by prothrombin time (PT) or international normalized ratio (INR) and activated partial thromboplastin time (aPTT) representing the intrinsic and extrinsic pathways of the clotting cascade, respectively. Although infrequently performed clinically, specific procoagulant factor levels can be measured. Fibrinolysis can generally be measured through fibrinogen levels, protein C and S levels, antithrombin III (ATIII) activity, euglobulin lysis time (ELT), and direct measurements of anticoagulant proteins (PAI-1, TAFI).1 There are also in vitro whole blood measures of the complete clotting system using thromboelastography (TEG), rotational thromboelastometry (ROTEM), and sonorheometry, which can give an overall assessment of the different stages of hemostasis, including the intrinsic fibrinolytic and antifibrinolytic pathways.2 In patients with cirrhosis every stage of hemostasis may be altered, putting them at risk for both bleeding and thrombosis. Classically, patients with cirrhosis have been thought of as “autoanticoagulated” because of an elevated INR and baseline thrombocytopenia owing to splenic sequestration and portal hypertension. Although routine tests such as platelet counts, INR, and aPTT may accurately reflect bleeding risk in general populations, multiple studies have shown that patients with cirrhosis have deficiencies in both the procoagulant and anticoagulant pathways, leading to a “rebalanced” coagulation system.3,4 In regard to primary hemostasis, patients with cirrhosis typically have a decreased number of circulating platelets. This is related to splenic sequestration, more rapid turnover and shorter half-life of platelets secondary to splenomegaly, and decreased production related to lower amounts of hepatic thrombopoetin.5,6 There may also be an immunologic component, although this appears to be a minor contributor.7 The decreased platelet count may be expected to increase the risk for bleeding, but patients with cirrhosis also have elevated levels of vWF, which increases in proportion to the severity of liver disease.8 In addition, there is a decrease in ADAMTS13, the vWF cleaving protein, and despite the underlying thrombocytopenia this increase in vWF may actually increase platelet adhesion.9,10 A study by Tripodi et al11 showed that in patients with cirrhosis a minimum platelet count of around 55×109/L is adequate for thrombin generation, allowing normal clot formation, and levels above 100×109/L have no increased benefit in primary hemostasis or in fibrin mesh formation when compared with controls. In some patients, these compensatory mechanisms may actually result in enhanced platelet adherence rather than dysfunctional primary hemostasis. In the second stage of hemostasis, clotting factors activate and begin to produce thrombin, predominantly on the surface of activated platelets, leading to subsequent deposition of the cross-linked fibrin mesh. In patients with cirrhosis, decreased levels of procoagulant proteins, such as factors II, V, VII, IX, X, and XI, suggest the potential for an anticoagulated state. In fact, 1 study showed a decrease in the activity of procoagulant proteins to as low as 20% to 46% in correlation with the severity of cirrhosis and portal hypertension.12 These deficiencies are easily seen in the standard clinical laboratory measurements of the INR and PT.

The C‐terminus of tissue factor pathway inhibitor‐α inhibits factor V activation by protecting the Arg1545 cleavage site

Background Factor V (FV) is a carrier and a cofactor of the anticoagulant protein tissue factor pathway inhibitor-α (TFPIα), whose basic C-terminus binds to an acidic region in the B-domain of FV. Proteolysis of FV at Arg709 , Arg1018 and Arg1545 by activated FX (FXa) or thrombin removes the B-domain, and converts FV into a procoagulant cofactor (activated FV [FVa]) of FXa in the prothrombinase complex. However, retention of the acidic region in partially activated FV makes prothrombinase activity susceptible to inhibition by TFPIα. Objective/Methods To investigate the effect of the TFPIα C-terminal peptide (TFPIα C-term) on thrombin generation in plasma and on FV activation in model systems. Results TFPIα C-term inhibited tissue factor-initiated and FXa-initiated thrombin generation in a dose-dependent manner. Failure to inhibit thrombin generation in FV-depleted plasma reconstituted with FVa indicated that the peptide effect was mediated by the acidic region of FV, and was localized at the level of FV activation and/or prothrombinase. In model systems, TFPIα C-term inhibited both FV activation and prothrombinase activity. Western blot analysis showed that the peptide impaired cleavage at Arg1545 by both thrombin and FXa. The inhibition was stronger for FV-short, which binds TFPIα with higher affinity. Similar results were obtained with full-length TFPIα. Conclusions Cleavage of FV at Arg1545 , which abolishes the anticoagulant properties of FV and commits FV to the procoagulant pathway, is inhibited by binding of the TFPIα C-terminus to the FV acidic region. Possible targets of this new anticoagulant function of TFPIα are low-abundance FV(a) species retaining the acidic region.

Factor VIIa Interaction with Endothelial Cell Protein C Receptor: Its Role in Hemostatic Effect of rFVIIa in Treating Hemophilia

Recent studies from our laboratory and others established that clotting factor FVII/FVIIa, which binds tissue factor and triggers the coagulation cascade, also binds endothelial cell protein C receptor (EPCR), a receptor that plays a critical role in protein C/activated protein C (APC)-mediated anticoagulation pathway. We postulated that FVIIa binding to EPCR might augment the hemostatic effect of rFVIIa in therapeutic conditions by down-regulating the EPCR-mediated anticoagulation pathway. Our recent studies showing the blockade of endogenous protein C binding to EPCR by administration of EPCR blocking antibodies augmented the hemostatic effect of rFVIIa in a mouse hemophilia model provided an indirect support to this hypothesis. However, studies from other investigators raised a possibility that FVIIa interaction with EPCR may influence the hemostatic effect of rFVIIa in vivo through EPCR-FVIIa directly activating FX or EPCR tethering FVIIa to provide an extended locale of procoagulant reactions on the endothelium. The present study is carried out to investigate the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of FVIIa in hemophilia therapy. The study employed wild-type, transgenic mice expressing no EPCR (EPCR-/-) or overexpressing EPCR (Tie2-EPCR). FVIII mAb (1 mg/kg) were given to mice to induce hemophilia. The saphenous vein incision bleeding model was used to evaluate the hemostatic effect of human rFVIIa. In this model, bleeding is induced by a sharp incision to the saphenous vein and the bleeding from the cut was monitored for 30 min. After each hemostasis incident, the clot was disrupted gently to reinitiate a new bleeding episode and average time to achieve hemostasis (ATH) was calculated.First, we investigated the role of EPCR in hemostasis by comparing the bleeding episodes in unchallenged wild-type, EPCR-/- and Tie2-EPCR mice following the saphenous vein incision. In this model, control wild-type mice had a median ATH of 55 sec (mean 63 ± 7 sec; n=9) and EPCR-/- mice had a median ATH of 67 sec (mean 74 ± 6 sec; n =12). The difference between them is not statistically significant. Surprisingly, the median ATH in EPCR overexpressing Tie2-EPCR mice was significantly lower (a median ATH of 43 sec; mean 46 ± 2 sec; n=10) compared to the ATH in wild-type or EPCR-/- mice. The reason for a shorter ATH observed in EPCR overexpressing mice is unknown at present. Analysis of blood count showed no significant differences among wild-type, EPCR-/- and Tie2-EPCR in their platelet count, RBC, and other blood cell count.Next, we induced acquired hemophilia condition in wild-type, EPCR-/- and Tie2-EPCR by injecting FVIII mAb (1 mg/kg) i.v. 2 h before inducing the bleeding. Administration of FVIII mAb markedly prolonged the bleeding time in wild-type and Tie2-EPCR mice (between 300 to 1800 sec) with a median ATH of 600 sec (mean 730 ± 277 sec) in wild-type mice and 750 sec (mean 939 ± 200 sec) in Tie2-EPCR mice. Interestingly, EPCR-/- mice were protected from the antibody-induced acquired hemophilia. Administration of FVIII mAb prolonged the bleeding time only marginally in this group of mice to a median ATH of 120 sec (mean 163 ± 38 sec). Administration of a low dose of rFVIIa (0.25 mg/kg) fully corrected the mild bleeding defect in EPCR-/- acquired hemophilia mice (median ATH of 67 sec; mean 86 ± 17 sec). Administration of 0.25 mg/kg rFVIIa is corrected the bleeding disorder only partially in wild-type acquired hemophilia mice (median ATH 164 sec; mean 303 ± 94 sec; n=5). Administration of either 1 or 4 mg/kg of rFVIIa was required to correct the bleeding in these mice (1 mg/kg dose: median ATH of 75 sec, mean 74 ± 1 sec; 4 mg/kg dose: median ATH of 37 sec; mean 40 ± 2 sec, n=5). In Tie2-EPCR hemophilia mice, administration of 1 mg/kg rFVIIa was not sufficient to fully achieve the hemostatic effect (median ATH 164 sec; mean 521 ± 237 sec; n=6). Administration of 4 mg/kg rFVIIa was required to restore the hemostasis in these mice (median ATH of 90 sec; mean 94 ± 7 sec, n= 6). In summary, the present data indicate that EPCR levels influence the hemostatic effect of rFVIIa in treating antibody-induced hemophilia. The present study rules out the possibility of direct activation of FX by FVIIa-EPCR complexes or EPCR tethering of FVIIa as probable mechanisms by which EPCR-FVIIa influences the hemostatic effect of rFVIIa. DisclosuresNo relevant conflicts of interest to declare.

A Novel Beneficial Role for Macitentan in Patients at High Thrombosis Risk: Attenuation of Platelet-Mediated Derangements of Blood Coagulation in Eisenmenger Syndrome

IntroductionEisenmenger syndrome is a rare manifestation of congenital cardiac disease with pulmonary hypertension. Potentially dangerous thrombotic and haemorrhagic complications frequently occur in patients with ES and raise significant clinical dilemmas. Underlying mechanisms are unknown but are thought to be linked to the underlying pulmonary vascular disease. Endothelin-1 (ET-1) signalling in the pulmonary vasculature is implicated in the pathogenesis of ES and Macitentan (a novel dual ET-1 receptor antagonist) has been shown to improve clinical outcomes. However the effect of Macitentan on reversing abnormal blood coagulation is unknown.We aimed to characterise derangements in physiological procoagulant and anticoagulant pathways in ES and to assess the effect of Macitentan therapy in modulating haemostasis in this disorder. We hypothesized that an enhanced understanding of pro-and anticoagulant mechanisms in these patients has the potential to improve risk stratification and targeting of antithrombotic therapy to patients at high risk of thrombosis.MethodsBlood was collected from subjects with ES and age/gender-matched healthy controls into citrated tubes, some containing corn trypsin inhibitor (CTI, a contact pathway inhibitor; final concentration 50µg/mL).Parameters of plasma thrombin generation in platelet-rich and platelet-poor plasma were assessed by calibrated automated thrombography (CAT) in the presence or absence of exogenous tissue factor (TF), anionic phospholipid (PL) and activated protein C (APC).ResultsPlatelet-dependent blood coagulation measured by CAT was enhanced in ES platelet-rich plasma (PRP) relative to healthy volunteers despite lower platelet counts: peak thrombin generation (TG) was significantly higher in ES subjects v controls in PRP (230.6±10.9 vs 188.3±13.1 nM; p=0.025) and lagtime to TG (6.1±0.3 vs 7.8±0.5 mins, p=0.005), time to peak TG (11.5±0.5 vs 15.2±0.6 mins, p=0.0003) and TG velocity index (43.9±3.7 vs 26.3±2.4 nM/min, p=0.001) were significantly accelerated. No significant differences in TG parameters in PPP were observed between groups suggesting a critical mechanistic role for platelet activity in supporting hypercoagulability in ES. TG in PRP was also found to be significantly enhanced in ES in the presence of CTI, suggesting that in vitro or in vivocontact pathway activation does not contribute to the enhanced coagulation activation observed in this disorder.The anticoagulant activity of APC is PL dependent and in healthy PPP incubated with exogenous PL (as a substitute for platelet PL), TG was potently inhibited. Conversely, in healthy PRP (in the absence of exogenous PL), APC activity was attenuated. Remarkably, this effect was not observed among the ES subjects, where APC anticoagulant activity was significantly increased in PRP, with significantly lower residual peak plasma thrombin generation and endogenous thrombin potential relative to that observed in healthy volunteer PRP. APC anticoagulant activity in PPP (both in the presence and absence of exogenous PL) was similar between groups. These findings suggest that platelets also modulate the activity of the APC pathway in ES, but in a manner which could promote bleeding.Among the 7 subjects who commenced macitentan therapy, at 6 months peak TG in PRP was significantly lower (234.5±9.8 vs 204.6±14.1 nM, p=0.002) and the enhanced sensitivity to APC in PRP was also attenuated. No significant difference in any parameter of TG was observed in PPP following macitentan therapy.ConclusionPlatelet activity appears to contribute significantly to hypercoagulability in ES but also appears to modulate the activity of endogenous anticoagulant pathways reflecting the clinical phenotype where bleeding and thrombotic risks frequently co-exist. Our findings suggest that targeted pharmacological agents such as the dual endothelin-1 antagonists (which crucially do not confer a bleeding risk) may attenuate platelet-mediated derangements in blood coagulation. Given the significant clinical dilemmas posed by the presence of competing thrombotic and haemorrhagic tendencies in ES, these finding are likely to be of significant translational relevance. DisclosuresWalsh:Actelion UK: Research Funding. Maguire:Actelion UK: Research Funding. Ní Áinle:Actelion UK: Research Funding.

Targeted Inhibition of Activated Protein C Anticoagulant Activity By Monoclonal Antibody HAPC1573 for Treatment of Hemophilia

In patients with hemophilia, hereditary defects in coagulation factors result in unstable clots and recurrent bleeding. Although the current standard of care focuses on replacement factor therapy, we propose that inhibition of the anticoagulant pathways may offer novel therapeutic opportunities. Activated protein C (APC) is one of the major anticoagulants, which works by degrading factors Va and VIIIa to maintain hemostatic balance. APC also exhibits cytoprotective effects, which include antiapoptotic effects, endothelial barrier protection, and anti-inflammatory effects. Antibodies are ideally suited to inhibit the anticoagulant activity of APC while preserving its cytoprotective activity.We have developed an anti-APC monoclonal antibody (mAb), HAPC1573 (murine immunoglubulin G1/kappa), using hybridoma technology, that specifically binds to human APC (hAPC) at 3 to 7 nM binding affinity (Kd) but not to its zymogen, protein C (PC) as determined by surface plasmon resonance (SPR) using a Biacore T200 instrument (GE Healthcare, Pittsburgh, PA). To investigate the binding epitope of mAb HAPC1573, APC was inhibited with Phe-Pro-Arg-chloromethylketone (PPACK). PPACK is an irreversible inhibitor of APC and forms a covalent bond with the catalytic triad Ser195 (chymotrypsin numbering).HAPC1573 bound comparably to PPACK-hAPC and untreated hAPC coated on an enzyme-linked immunosorbent assay (ELISA) plate, suggesting that the binding epitope of HAPC1573 is located outside the active site of APC. These ELISA results were also confirmed by SPR analyses. HAPC1573 inhibited the cleavage of a small peptide substrate Spectrozyme PCa (Sekisui Diagnostics, Lexington, MA) by hAPC up to 40%. The antibody protected factors Va and VIIIa from APC-mediated inactivation in a dose-dependent manner. HAPC1573 significantly reduced activated partial thromboplastin time of hemophilic plasma and enhanced thrombin generation (assessed by thrombin generation assay) in the presence of thrombomodulin. HAPC1573 inhibited the anticoagulant activity of APC without affecting its cytoprotective functions, as measured by histone-mediated cytotoxicity assays on human umbilical vein endothelial cells (up to 300 nM HAPC1573). Given its cross-reactivity with monkey APC, the antibody was evaluated in Cynomolgus monkeys for therapeutic efficacy and safety. Intravenous administration of the antibody at 3 and 10 mg/kg significantly shortened bleeding time after injury and restored hemostasis in a dose-dependent manner in an anti-FVIII antibody-induced hemophilia monkey model (Figure). Administration of a sheep-anti-FVIII antibody (Haematologic Technologies, Inc., Essex Junction, VT) reduced plasma FVIIIa activity to below the lower limit of quantification (LLOQ; Figure [left panel]) and led to a significantly longer bleeding time in normal monkeys (Figure; right panel), recapitulating the hemophilia A phenotype. This prolonged bleeding time was partially reduced by 270 µg/kg of recombinant factor VIIa (rFVIIa; NovoSeven, Novo Nordisk, Plainsboro, NJ) used as a positive control for these studies. There was a statistically significant dose-dependent reduction in bleeding time by HAPC1573 with the 10-mg/kg dose restoring the bleeding time back to normal.This study provides in vivo proof of concept of using anti-APC antibody for hemophilia. HAPC1573 represents an anti-APC antibody with therapeutic utility for patients with hemophilia with inhibitors. [Display omitted] DisclosuresZhao:Bayer Pharmaceuticals: Employment. Yegneswaran:Bayer Healthcare Pharmaceuticals: Employment. Sim:Bayer: Employment. Patel:Bayer Pharmaceuticals: Employment. Schneider:Bayer HealthCare LLC: Employment, Patents & Royalties. McLean:Bayer: Employment, Equity Ownership. Zhu:Bayer Healthcare: Employment. Jiang:Bayer Pharmaceuticals: Employment. Gu:Bayer Pharmaceuticals: Employment. Ivens:Bayer Pharmaceuticals: Employment. Xu:Shanghai RAAS Blood Products Co.Lt: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: Patent. Bringmann:Bayer Corporation, Parmaceuticals Division: Employment. Kauser:Bayer: Employment.

Thrombin generation and cell‐dependent hypercoagulability in sickle cell disease

Introduction 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.

Ischemic Colitis Complicated by Consumptive Coagulopathy

Colonic ischemia-induced reperfusion injury can lead to multisystem organ failure, regardless of ischemic colitis etiology. However, it rarely leads to consumptive coagulopathy. A review of the current literature shows that there have been limited reported cases of colonic ischemia-induced consumptive coagulopathy. Known etiologies of disseminated intravascular coagulopathy (DIC) include sepsis, malignancy, trauma, and intravascular hemolysis. Ischemic colitis is not a known cause of disseminated intravascular coagulopathy; however, we can relate it to the mechanism of sepsis-induced disseminated intravascular coagulopathy as sepsis is frequently complicated by disseminated intravascular coagulopathy. Disseminating intravascular coagulopathy is the activation of intravascular coagulation culminating in intravascular fibrin formation and deposition into the microvasculature. Secondary fibrinolysis accompanies coagulation activation. Intravascular deposition of fibrin in sepsis leads to a diffuse obstruction of the microvascular bed resulting in progressive organ dysfunction, such as in acute respiratory distress syndrome, acute kidney injury, hypotension, and shock liver. Both the coagulation activation and the consumption of coagulation inhibitors leading to anticoagulation pathways are activated in sepsis, leading to consumptive coagulopathy. Consumptive coagulopathy was an unexpected response to ischemic colitis. We present a case of a 71 year old Hispanic female with past medical history significant for hypertension presented to our facility with sudden onset diffuse abdominal pain and bright red blood per rectum. Work up revealed hematologic profile significant for DIC in addition to acute kidney injury and ischemic hepatitis, negative for schistocytes on peripheral blood smear. Colonoscopy revealed severe ischemic colitis with submucosal hemorrhage and necrosis involving descending colon. Biopsy from colonoscopy was consistent with observation, revealing acute colitis with focal ischemic changes, granulation tissue, and acute inflammatory exudate of descending colon. We believe that the intense inflammatory reaction of ischemic colitis led to this consumptive coagulopathy. While the known etiologies of disseminated intravascular coagulopathy are numerous, ischemic colitis is rarely presented as one.Table 1: Initial labsTable 2: Resolution of disseminated intravascular coagulopathy in a case of ischemic colitisFigure 1Figure 2

Genetic Risk Factors of Venous Thromboembolism in the East Algerian Population.

Many genetic risk factors have been identified for causing venous thromboembolism (VTE). Most of them affect the function of natural anticoagulant pathways, particularly the protein C system, although recent studies suggest a role of components of the hematopoietic pathway in the etiology of venous thrombosis. In this case-control study, we aimed to determine the frequency of prothrombin G20210A and factor V Leiden (FVL) G1691A polymorphisms and protein C, protein S, and antithrombin III deficiencies in the East Algerian population and to investigate whether these genetic factors are associated with VTE. On the other hand, our study tends to evaluate the status of JAK2V617F and calreticulin (CALR) mutations among these cases. The participants consisted of 121 cases with VTE and 146 healthy controls. Polymorphisms of FVL G1691A and prothrombin G20210A were genotyped by polymerase chain reaction (PCR) restriction fragment length polymorphism. JAK2-V617F and calreticulin mutations were analyzed by quantitative PCR and PCR followed by capillary electrophoresis sequencing, respectively. Protein C, protein S, and antithrombin levels were determined and then hereditary deficiencies were identified. Of all cases and controls, none was a carrier of the antithrombin III deficiency, prothrombin gene G20210A, and CALR mutations. Only 1 case reported having a positive JAK2 mutation (mutant allele burden was 15%). The FVL mutation (GA/AA) was found in 14 (11.6%) cases and 2 (1.4%) controls and it was significantly different between both the groups ( P = .001). Deficiencies of protein S and protein C were detected in 17 (18.8%) cases. The univariate analysis resulted in a significant impact of FVL (odds ratio [OR] = 9.4, 95% confidence interval [CI] = 2.1-42.3; P = .003) and of protein S deficiency (OR = 16.9, 95% CI =2.1-132.8, P = .007) on the VTE status. Both factors stayed significant after adjustment for sex and age. The OR of the protein C deficiency was slightly elevated (OR = 6.4, 95% CI = 0.7-55.5), but it did not reach the level of statistical significance ( P = .091), and it was therefore not considered as a risk factor. In conclusion, coagulant factor V gene G1691A mutation and protein S deficiency constitute important genetic risk factors in patients with VTE in Eastern Algeria. The somatic mutation of JAK2 V617F and CALR mutations are less frequent causes of VTE, thus routine testing for these mutations is not recommended.

Decreased Activity of the Protein C Anticoagulant Pathway in the Early Hours of Paroxysmal Atrial Fibrillation.

Increased coagulation activity has been established in paroxysmal atrial fibrillation (PAF), but data on the anticoagulant system are scarce. To examine the protein C anticoagulant pathway in the early hours of the disease. Fifty-one patients (26 men and 25 women; mean age 59.84 ± 1.60 years) and 52 controls (26 men and 26 women; mean age 59.50 ± 1.46 years) were selected for the study. Protein C antigen and its activity, total protein S, free protein S and its activity, soluble forms of endothelial protein C receptor (sEPCR), and thrombomodulin (sTM) were examined in the plasma. The indicators were studied in patients between the 2nd and the 24th hour after the onset of arrhythmia. Levels of protein C were significantly elevated in patients compared to controls (111.40% ± 6.66% vs 94.83% ± 4.47%; P = .039). Protein C activity showed significant reduction in PAF (73.13% ± 5.80% vs 103.3% ± 3.80%; P < .001). Total protein S levels did not differ significantly (108.20% ± 4.07% vs 102.40% ± 3.65%; P = .30). Free protein S (76.81% ± 6.01% vs 122.10% ± 3.97%; P < .001) and its activity (71.39% ± 6.27% vs 119.50% ± 6.54%; P < .001) were reduced in patients. Higher levels of sEPCR (203.10 ± 10.33 vs 133.10 ± 7.37 ng/mL; P < .001) and sTM (6.50±0.40 vs 4.48±0.28 ng/mL; P < .001) were measured in PAF. Protein C activity is reduced still in the first hours (until the 24th hour) of PAF clinical manifestation, determining reduced activity of the anticoagulant pathway as a whole. The established low levels of free protein S and its activity as well as low sEPCR and sTM levels are a possible explanation of the changes in protein C activity.

Disseminated intravascular coagulation.

Disseminated intravascular coagulation (DIC) is an acquired syndrome characterized by widespread intravascular activation of coagulation that can be caused by infectious insults (such as sepsis) and non-infectious insults (such as trauma). The main pathophysiological mechanisms of DIC are inflammatory cytokine-initiated activation of tissue factor-dependent coagulation, insufficient control of anticoagulant pathways and plasminogen activator inhibitor 1-mediated suppression of fibrinolysis. Together, these changes give rise to endothelial dysfunction and microvascular thrombosis, which can cause organ dysfunction and seriously affect patient prognosis. Recent observations have pointed to an important role for extracellular DNA and DNA-binding proteins, such as histones, in the pathogenesis of DIC. The International Society on Thrombosis and Haemostasis (ISTH) established a DIC diagnostic scoring system consisting of global haemostatic test parameters. This scoring system has now been well validated in diverse clinical settings. The theoretical cornerstone of DIC management is the specific and vigorous treatment of the underlying conditions, and DIC should be simultaneously managed to improve patient outcomes. The ISTH guidance for the treatment of DIC recommends treatment strategies that are based on current evidence. In this Primer, we provide an updated overview of the pathophysiology, diagnosis and management of DIC and discuss the future directions of basic and clinical research in this field.

Innovative Pharmacological Therapies for the Hemophilias Not Based on Deficient Factor Replacement.

In recent years, advances in the pharmacological treatment of hemophilias A and B have mainly focused on the development of long-acting factor (F)VIII and FIX products. Alternative approaches not based on the replacement of the missing factor have also been explored, with the aim of producing therapeutic agents with reduced immunogenicity and yet equally effective in patients with or without inhibitors. These new classes of hemostatic agents act mainly by bypassing the need of FVIII and FIX in tenase formation, quenching anticoagulant pathways, enhancing the activity of some coagulation factors or stabilizing the fibrin clot. Current knowledge on the status of development of these novel molecules is summarized in this narrative review. We also surmise that the main interests for these products not based on the replacement of FVIII or FIX in deficient patients pertain to the potential for bleeding prevention in inhibitor patients, an earlier and easier prophylaxis implementation thanks to subcutaneous administration and prolonged half-life, and a low immunogenicity with the potential for prevention of inhibitor development in high-risk patients.

Targeting TFPI for hemophilia treatment

Hemophilia is a severe bleeding disorder treated by infusion of the missing blood coagulation protein, factor VIII or factor IX. The discovery and characterization of the anticoagulant protein tissue factor pathway inhibitor (TFPI) led to the realization that inhibition of TFPI activity could restore functional hemostasis through the extrinsic blood coagulation pathway in a manner that does not require the activity of factors VIII or IX. There are currently several therapeutic agents that inhibit TFPI in development for treatment of hemophilia. A comprehensive understanding of TFPI structure, biochemistry, and cellular expression is necessary to understand how it modulates bleeding in hemophilia and the physiological impact of therapeutic agents targeting TFPI.