Vascular Endothelial Cells Produce Coagulation Factors That Control Their Growth via Joint Protease-Activated Receptor and C5a Receptor 1 (CD88) Signaling.

As per the classical view of the coagulation system, it functions solely in plasma to maintain hemostasis. An experimental approach modeling vascular reconstitution was used to show that vascular endothelial cells (ECs) endogenously synthesize coagulation factors during angiogenesis. Intracellular thrombin generated from this synthesis promotes the mitotic function of vascular endothelial cell growth factor A (VEGF-A). The thrombin concurrently cleaves C5a from EC-synthesized complement component C5 and unmasks the tethered ligand for EC-expressed protease-activated receptor 4 (PAR4). The two ligands jointly trigger EC C5a receptor-1 (C5ar1) and PAR4 signaling, which together promote VEGF receptor 2 growth signaling. C5ar1 is functionally associated with PAR4, enabling C5a or thrombin to elicit Gαi and/or Gαq signaling. EC coagulation factor and EC complement component synthesis concurrently down-regulate with contact inhibition. The connection of these processes with VEGF receptor 2 signaling provides new insights into mechanisms underlying angiogenesis. Knowledge of endogenous coagulation factor/complement component synthesis and joint PAR4/C5ar1 signaling could be applied to other cell types.

Synthesis of coagulation factors during long-term ex situ liver perfusion.

Robust viability assessment of grafts during normothermic liver perfusion is a prerequisite for organ use. Coagulation parameters are used commonly for liver assessment in patients. However, they are not yet included in viability assessment during ex situ perfusion. In this study, we analysed coagulation parameters during one week ex situ perfusion at 34℃. Eight discarded human livers were perfused with blood-based, heparinised perfusate for one week; perfusions in a further four livers were terminated on day 4 due to massive ongoing cell death. Coagulation parameters were well below the physiologic range at perfusion start. Physiologic levels were achieved within the first two perfusion days for factor V (68.5 ± 35.5%), factor VII (83.5 ± 26.2%), fibrinogen (2.1 ± 0.4 g/L) and antithrombin (107 ± 26.5%) in the livers perfused for one week. Despite the increased production of coagulation factors, INR was detectable only at 24h of perfusion (2.1 ± 0.3) and prolonged thereafter (INR > 9). The prolongation of INR was related to the high heparin level in the perfusate (anti-FXa > 3 U/mL). Intriguingly, livers with ongoing massive cell death also disclosed synthesis of factor V and improved INR. In summary, perfused livers were able to produce coagulation factors at a physiological level ex situ. We propose that single coagulation factor analysis is more reliable for assessing the synthetic function of perfused livers as compared to INR when using a heparinised perfusate.

Engineering CRISPR/Cas9 for Multiplexed Recombinant Coagulation Factor Production.

Current hemostatic agents are obtained from pooled plasma from multiple donors requiring costly pathogen screening and processing. Recombinant DNA-based production represents an engineering solution that could improve supply, uniformity, and safety. Current approaches are typically for single gene candidate peptides and often employ non-human cells. We devised an approach where multiple gene products could be produced from a single population of cells. We identified gene specific Synergistic Activation Mediators (SAM) from the CRISPR/Cas9 system for targeted overexpression of coagulation factors II, VII, IX, X, and fibrinogen. The components of the CRISPR-SAM system were expressed in Human Embryonic Kidney Cells (HEK293), and single (singleplex) or multi-gene (multiplex) upregulation was assessed by quantitative RT-PCR (qRT-PCR) and protein expression by ELISA analysis. Factor II, VII, IX, and X singleplex and multiplex activation resulted in 120-4700-fold and 60-680-fold increases in gene expression, respectively. Fibrinogen sub-unit gene activation resulted in a 1700-92,000-fold increases and 80-5500-fold increases in singleplex or multiplex approaches, respectively. ELISA analysis showed a concomitant upregulation of candidate gene products. Our findings demonstrate the capability of CRISPR/Cas9 SAMs for single or multi-agent production in human cells and represent an engineering advance that augments current recombinant peptide production techniques.

Leptin induces the generation of procoagulant, tissue factor bearing microparticles by human peripheral blood mononuclear cells.

BACKGROUND: Obesity is linked to increased thrombotic risk. Circulating leptin concentration correlates with body mass index. Microparticles are small (.05-1 µm) vesicles shed by activated and apoptotic cells, involved in numerous pathophysiologically relevant phenomena including blood coagulation and thrombosis. We tested the hypothesis that leptin induces the shedding of procoagulant, tissue factor bearing microparticles by human peripheral blood mononuclear cells, and investigated the intracellular mechanisms leading to microparticle release upon incubation with leptin. METHODS: Peripheral blood mononuclear cells were isolated from healthy donors. Cells were incubated with leptin in the presence or in the absence of a phospholipase C inhibitor, U73122, a calmodulin inhibitor, W-7, and three inhibitors of mitogen activated protein kinases. Microparticle generation was assessed as phosphatidylserine concentration with a prothrombinase assay and by cytofluorimetric analysis. Tissue factor expression on microparticles was measured with a one-stage clotting assay. Intracellular calcium concentration was assessed by a fluorescent probe. RESULTS: Leptin increased intracellular calcium mobilization and stimulated the generation of tissue factor-bearing MP by peripheral blood mononuclear cells, as assessed by phosphatidylserine quantification, clotting tests and flow-cytometry. U73122, PD98059 (an extracellular signal-regulated kinase1/2 inhibitor), and W-7, significantly inhibited leptin-induced MP release. SB203580 (a p38 inhibitor), and SP600125 (a c-Jun N-terminal kinase inhibitor) had no effect. CONCLUSION: Leptin-induced generation of procoagulant microparticles might represent a link between obesity and atherothrombotic risk. Inhibition of leptin-induced microparticle generation might prove a viable strategy for the reduction of such risk in obese individuals.

Treatment of Coagulopathy Related to Hepatic Insufficiency.

OBJECTIVES: To provide a concise review of the medical management of coagulopathy related to hepatic insufficiency. This review will focus on prevention and management of bleeding episodes in patients with hepatic insufficiency. The treatment and prevention of thromboembolic complications will also be addressed. DATA SOURCES: Electronic search of PubMed database using relevant search terms, including hepatic coagulopathy, hemorrhage, liver diseases, blood coagulation disorders, blood transfusion, disseminated intravascular coagulation, and liver failure. Subsequent searches were done on specific issues. STUDY SELECTION: Articles considered include original articles, review articles, guidelines, consensus statements, and conference proceedings. DATA EXTRACTION: A detailed review of scientific, peer-reviewed data was performed. Relevant publications were included and summarized. DATA SYNTHESIS: Available evidence is used to describe and summarize currently available tests of hemostasis, utilization of prohemostatic agents, transfusion strategies, use of prophylactic anticoagulation and treatment of thromboembolic events in patients with hepatic insufficiency. CONCLUSIONS: Dynamic changes to hemostasis occur in patients with hepatic insufficiency. Routine laboratory tests of hemostasis are unable to reflect these changes and should not be used exclusively to evaluate coagulopathy. Newer testing methods are available to provide data on the entire spectrum of clotting but are not validated in acute bleeding. Prohemostatic agents utilized to prevent bleeding should only be considered when the risk of bleeding outweighs the risk of thrombotic complications. Restrictive transfusion strategies may avoid exacerbation of acute bleeding. Prophylaxis against and treatment of thromboembolic events are necessary and should consider patient specific factors.

Limited ability to activate protein C confers left atrial endocardium a thrombogenic phenotype: a role in cardioembolic stroke?

BACKGROUND AND PURPOSE: Atrial fibrillation is the most important risk factor for cardioembolic stroke. Thrombi form in the left atrial appendage rather than in the right. The causes of this different thrombogenicity are not well-understood. The goal herein was to compare the activation of the anticoagulant protein C and the thrombomodulin and endothelial protein C receptor/activated protein C receptor expression on the endocardium between right and left atria. METHODS: We harvested the atria of 6 monkeys (Macaca fascicularis) and quantified their ability to activate protein C ex vivo and we measured the thrombomodulin and endothelial protein C receptor expression by immunofluorescence. RESULTS: We found the ability to activate protein C decreased by half (P=0.028) and there was lower expression of thrombomodulin in the left atrial endocardium than the right (52.5±19.9 and 72.1±18.8 arbitrary intensity units, mean±standard deviation; P=0.028). No differences were detected in endothelial protein C receptor expression. CONCLUSIONS: Impaired protein C activation on the left atrial endocardium attributable to low thrombomodulin expression may explain its higher thrombogenicity and play a role in cardioembolic stroke.

The spectrum of coagulation abnormalities in thyroid disorders.

The hemostatic balance is a complex system where the delicate equilibrium is regulated by several factors including hormones. A variety of endocrine disorders have been reported to be associated with coagulation abnormalities, ranging from mild laboratory changes to clinically relevant thrombotic or bleeding manifestations. In this review, we summarize the current knowledge on the main abnormalities of the coagulation and fibrinolytic systems associated with thyroid dysfunctions. Overall, although mostly based on uncontrolled studies, data in the literature suggest that patients with hyperthyroidism or subclinical hypothyroidism have a hypercoagulative state, whereas patients with overt hypothyroidism have a bleeding tendency.

Endothelial cell protein C receptor cellular localization and trafficking: potential functional implications.

Although the binding of endothelial cell protein C receptor (EPCR) to its ligands is well characterized at the biochemical level, it remains unclear how EPCR interaction with its ligands at the cell surface impacts its cellular trafficking. We characterized the cellular localization and trafficking of EPCR in endothelial cells and a heterologous expression system. Immunofluorescence confocal microscopy studies revealed that a majority of EPCR is localized on the cell surface in membrane microdomains that are positive for caveolin-1. A small fraction of EPCR is also localized intracellularly in the recycling compartment. Factor VIIa (FVIIa) or activated protein C binding to EPCR promoted the internalization of EPCR. EPCR and EPCR-bound ligands were endocytosed rapidly via a dynamin- and caveolar-dependent pathway. The endocytosed receptor-ligand complexes were accumulated in a recycling compartment before being targeted back to the cell surface. EPCR-mediated FVIIa endocytosis/recycling also resulted in transport of FVIIa from the apical to the basal side. In vivo studies in mice showed that blockade of EPCR with EPCR-blocking antibodies impaired the early phase of FVIIa clearance. Overall, our results show that FVIIa or activated protein C binding to EPCR promotes EPCR endocytosis, and EPCR-mediated endocytosis may facilitate the transcytosis of FVIIa and its clearance from the circulation.

Recombinant plasma proteins.

For almost 50 years, the fractionation of human plasma has been the sole possible source of a wide range of therapeutic proteins--such as coagulation factors, anticoagulants, immunoglobulins, and albumin--essential to the treatment of serious congenital or acquired bleeding or immunological diseases. In the last 20 years, the application of recombinant technologies to mammalian cell cultures has made possible--although with some limitations in productivity, costs, and immunogenic risks--to produce and commercialize complex plasma glycoproteins for human therapeutic applications and has opened the way to the development of new molecular entities. Today, the advanced exploration of alternative cell lines and enhanced cell culture systems, as well as the development of alternative expression technologies, such as transgenic animals, is opening a new era in the production of the full range of recombinant plasma protein therapeutics. In this review, we examine the achievements and ongoing challenges of the recombinant DNA technology as a platform for the production of plasma proteins and the advantages and limitations of such products compared to fractionated plasma proteins.

Egr-1, a master switch coordinating upregulation of divergent gene families underlying ischemic stress.

Activation of the zinc-finger transcription factor early growth response (Egr)-1, initially linked to developmental processes, is shown here to function as a master switch activated by ischemia to trigger expression of pivotal regulators of inflammation, coagulation and vascular hyperpermeability. Chemokine, adhesion receptor, procoagulant and permeability-related genes are coordinately upregulated by rapid ischemia-mediated activation of Egr-1. Deletion of the gene encoding Egr-1 strikingly diminished expression of these mediators of vascular injury in a murine model of lung ischemia/reperfusion, and enhanced animal survival and organ function. Rapid activation of Egr-1 in response to oxygen deprivation primes the vasculature for dysfunction manifest during reperfusion. These studies define a central and unifying role for Egr-1 activation in the pathogenesis of ischemic tissue damage.

Hormones of hypothalamic-pituitary-thyroid axis are significant regulators of synthesis and secretion of vitamin K-dependent plasma coagulation factors.

Present data about hormonal regulation of haemostasis are often contradictory and are mostly based on clinical observations. The aim of the current research is to study the effects of the hormones of hypothalamic-pituitary-thyroid (HPT) axis on plasma levels (i.e. on the synthesis and secretion) of vitamin K-dependent coagulation factors in rats. The study was carried out on 65 male Wistar rats, divided into five groups. The animals were injected subcutaneously (s.c.) once daily for three consecutive days as follows: the first group was injected with Thyrotropin releasing hormone (TRH), in a dose of 0.06 mg/kg b.w.; the second group by Thyroid stimulating hormone (TSH), with a dose of 1 MU/kg b.w., the third and the fourth group respectively with Liothyroninum (Triiodothyronin ? T3) and Levothyroxinum (Thyroxin ? T4) with a dose of 0.08 mg/kg b.w. each. The control group rats were injected with saline (the solvent of the hormones), following the same schedule and volume per kg b.w. The necessary quantity of blood was acquired by a cardiac puncture under ether narcosis, and antigen levels of plasma factors II, VII, IX and X (FII:Ag, FVII:Ag, FIX:Ag and FX:Ag) were determined by ELISA kits (Diagnostica Stago, France). TRH, TSH, T3 and T4 significantly decreased the plasma antigen levels of FII and FVII (p<0.001). TRH, T3 and TSH reduced significantly FIX:Ag level( p<0.001 for TRH and T3 and p<0.05 for TSH) while T4 did not exert significant changes ( p>0.05). FX:Ag level was also significantly reduced by TRH, T3 (p<0.001), TSH and T4 (p<0.01). Plasma levels of vitamin K-dependent coagulation factors FІІ:Ag, FVІІ:Ag, FІХ:Ag and FХ:Ag are significantly reduced under the influence of the hormones of hypothalamic-pituitary-thyroid axis which signifies their decreased synthesis and secretion. T4 does not induce substantial changes in FIX:Ag plasma level.

Human endothelial cells and fibroblasts express and produce the coagulation proteins necessary for thrombin generation.

In a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV-FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin-antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and γ-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin.

Induction of monocyte procoagulant activity by murine hepatitis virus type 3 parallels disease susceptibility in mice.

The in vitro induction of procoagulant activity (PCA) in murine peripheral blood mononuclear cells (PBM) by murine hepatitis virus type 3 (MHV-3) correlates with the disease susceptibility in three strains of mice. PBM from BALB/c mice, a strain in which MHV-3 infection results in fatal acute fulminant hepatitis, responds to the virus with a robust PCA response, whereas PBM from C3H/St mice, a strain which develops mild acute hepatitis followed by chronic hepatitis, only exhibit a modest PCA response. In contrast, PBM from A/J mice, a strain fully resistant to MHV-3, generate no increase in PCA above control levels. The induction phase of MHV-3 PCA is rapid, with an increase within 1-1.5 h, with maximum activity at 18h, and it precedes MHV-3 replication in either 17 CL1 cells, a fully permissive cell line, or in monocytes from these strains of mice. The PCA response of BALB/c PBM exceeds the response to any other known stimulus. No induction occurs upon direct stimulation of monocytes by MHV-3, but in the presence of lymphocyte collaboration, the PCA response is observed first at a lymphocyte:monocyte ratio of 2:1 and reaches a maximum as the lymphocyte:monocyte ratio approaches 4:1. This response appears to provide a functional marker for susceptibility to MHV-3 infection in inbred strains of mice and could be important in the pathogenesis of MHV-3-induced disease.

Induction and detection of a human endothelial activation antigen in vivo.

We used a murine mAb, H4/18, raised by immunization with IL-1-treated human umbilical vein endothelial cell cultures, to localize an endothelial activation antigen in induced human delayed hypersensitivity reactions (DHR) and in pathological tissues. We used streptococcus varidase to elicit DHR in human skin and we examined sequential skin biopsies with the immunoperoxidase technique. There was no staining for H4/18 binding antigen in normal endothelium of skin and other tissues; strong positive staining, localized to vascular endothelium, was seen at 16 and 23 h but disappeared by 6 d, when the DHR had faded. H4/18 binding antigen, also confined to endothelium, was detected in lymph nodes, skin, and other tissues exhibiting immune/inflammatory reactions. The studies indicate that H4/18 is a useful marker for activated endothelium in vivo and they support the relevance of in vitro studies on inducible endothelial cell functions.

Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration.

Systemic infusion of low concentrations of tumor necrosis factor/cachectin (TNF) into mice that bear TNF-sensitive tumors leads to activation of coagulation, fibrin formation, and occlusive thrombosis exclusively within the tumor vascular bed. To identify mechanisms underlying the localization of this vascular procoagulant response, a tumor-derived polypeptide has been purified to homogeneity from supernatants of murine methylcholanthrene A-induced fibrosarcomas that induces endothelial tissue factor synthesis and expression (half-maximal response at approximately 300 pM), and augments the procoagulant response to TNF in a synergistic fashion. This tumor-derived polypeptide was identified as the murine homologue of vascular permeability factor (VPF) based on similar mobility on SDS-PAGE, an homologous NH2-terminal amino acid sequence, and recognition by a monospecific antibody to guinea pig VPF. In addition, VPF was shown to induce monocyte activation, as evidenced by expression of tissue factor. Finally, VPF was shown to induce monocyte chemotaxis across collagen membranes and endothelial cell monolayers. Taken together, these results indicate that VPF can modulate the coagulant properties of endothelium and monocytes, and can promote monocyte migration into the tumor bed. This suggests one mechanism through which tumor-derived mediators can alter properties of the vessel wall.

Haemostasis and thrombosis in liver disease.

Liver disease impacts on both primary and secondary haemostatic mechanisms and historically these changes were thought to underpin the bleeding diathesis. However, bleeding complications in patients with liver disease are unpredictable, with the majority of haemorrhagic episodes occurring as a result of porto-systemic varices. Thrombosis is an increasingly recognised complication and systemic hypercoagulability may contribute to the development of parenchymal extinction and accelerated hepatic fibrosis. Routine laboratory tests do not reliably predict the risk of haemorrhage and the optimal management strategy to avert potential bleeding complications is yet to be established. There may be a future role for global coagulation assays, such as thrombelastography and thrombin generation, in both stratifying the risk of bleeding and guiding management of these patients.

Effect of preoperative biliary drainage on coagulation and fibrinolysis in severe obstructive cholestasis.

GOALS: To evaluate the function of coagulation and fibrinolysis in cholestatic patients before and after preoperative biliary drainage (PBD). BACKGROUND: Cholestasis owing to an obstructive biliary malignancy is associated with postoperative complications related to a proinflammatory state, an impaired hepatic synthesis function, and a potential derangement of hemostasis. Hence, PBD is advocated for cholestatic patients undergoing major surgery. STUDY: Plasma coagulation and fibrinolytic parameters were assessed in 24 cholestatic patients and 10 controls. In 9 cholestatic patients, the parameters were reassessed at least 4 weeks after PBD. RESULTS: Compared with controls, cholestatic patients showed lower concentrations (P<0.001) of plasma vitamin K-dependent factors II and VII, whereas prothrombin time, activated partial thromboplastin time, and factor V were unaltered. Thrombin generation was increased in cholestatic patients, as reflected by higher plasma concentrations of thrombin-antithrombin complexes and D-dimers. Fibrinolysis was significantly impaired as evidenced by low plasminogen activator activity (PAA) owing to an increase in plasminogen activator inhibitor -1). Elevated markers for thrombin generation thrombin-antithrombin decreased after PBD from 10.7±1.2 to 5.7±0.7 ng/mL (P<0.05). Additionally, impairment of fibrinolysis in cholestatic patients resolved after PBD (plasminogen activator inhibitor-1 levels decreased from 19±1 to 10±1 IU/mL and plasminogen activator activity increased from 82±3 to 110±4%, respectively). D-dimers remained unaltered after PBD, likely because of normalization of coagulation and fibrinolytic activity. CONCLUSIONS: Obstructive cholestasis is associated with a procoagulant state, despite an impaired vitamin K-dependent coagulation factor synthesis. Virtually all alterations in coagulation and fibrinolysis were reversed by biliary drainage.

New synthesis of a platelet-specific protein: platelet factor 4 synthesis in a megakaryocyte-enriched rabbit bone marrow culture system.

The site of synthesis of platelet-specific proteins remains to be established. With the use of short-term megakaryocyte-enriched cultures, direct evidence was obtained to show that megakaryocytes synthesize the platelet-specific protein, platelet factor 4. A megakaryocyte-enriched fraction of rabbit bone marrow for culture was obtained by centrifugal elutriation and cultured with [3H]leucine. Newly synthesized 3H-platelet factor 4 was sought by copurification with added carrier rabbit platelet factor 4, using heparin agarose affinity chromatography and immunoprecipitation with specific goat anti-rabbit platelet factor 4 antisera. SDS PAGE of the washed immunoprecipitates demonstrated a [3H]leucine-containing peak which migrated identically with purified homogeneous rabbit platelet factor 4. A second, slightly larger molecular-weight protein was identified in the gels also, suggesting that rabbit platelet factor 4 may be synthesized as a larger molecular-weight precursor in rabbit megakaryocytes. These results provide direct evidence that the platelet-specific protein, platelet factor 4, is synthesized in rabbit megakaryocytes before it is packaged into alpha-granules for release in circulating platelets.

Biosynthesis of von Willebrand protein by human endothelial cells: processing steps and their intracellular localization.

Biosynthesis of von Willebrand protein by human umbilical vein endothelial cells involved distinct processing steps marked by the presence of several intermediate molecular species. Examination of endoglycosidase H sensitivity of these intracellular intermediates indicated that the processing steps occurred in at least two separate cellular compartments. In the pre-Golgi apparatus (most probably the endoplasmic reticulum), the high mannose carbohydrates were added onto the precursor monomer chains and the 260,000-mol-wt monomers dimerized by interchain disulfide bond formation. The other processing steps have been localized to the Golgi apparatus and later compartments (e.g., Weibel-Palade bodies). High mannose carbohydrate was converted to the complex type, leading to the appearance of a larger precursor subunit of 275,000 mol wt. The 275,000-mol-wt species was not formed if carbohydrate processing was inhibited by the ionophore monensin. From the large pool of dimers of precursor subunits, the high molecular weight multimers were built. These dimer molecules appeared to have free sulfhydryls which might have been involved in the interdimer disulfide bond formation. Simultaneously with multimerization, the precursor subunits were cleaved to the 220,000-mol-wt form. The cleavage of the pro-sequence was not likely to be an absolute requirement for von Willebrand protein multimerization or secretion, as the 275,000-mol-wt precursor subunit was present in secreted high molecular weight multimers of the protein.