Comparative Transcriptomic Analyses of Three Species of Placobdella (Rhynchobdellida: Glossiphoniidae) Confirms a Single Origin of Blood Feeding in Leeches.

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar -this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

Novel Functions of the Anticoagulant Activated Protein C in Maintaining Skin Barrier Integrity to Impact on Skin Disease.

The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. When the barrier is disrupted any of a number of diseases, such as chronic wounds, psoriasis, pemphigus, atopic dermatitis or toxic epidermal necrolysis, can take hold. Activated protein C (APC) or its precursor, protein C, is abundantly expressed by skin epidermal keratinocytes and stimulates their proliferation and migration, and inhibits apoptosis and inflammation, leading to a healing phenotype. Importantly, APC also increases the barrier function of keratinocytes by promoting expression and cell-cell contact redistribution of tight junction proteins. These cytoprotective properties of APC on epidermal keratinocytes place it as an exciting new therapy for skin disorders associated with the disruption of barrier function and inflammation.

[New oral anticoagulant pharmacology]. / Améliorations pharmacologiques apportées par les nouveaux anticoagulants oraux.

All new oral anticoagulants are direct specific reversible inhibitors, either direct factor Xa inhibitors or inhibitors of thrombin. The pharmacokinetic of the new drugs is mediated by P- glycoprotein (P-gp) and metabolised by liver enzymes for some of them, principally cytochrome P450. That explains an important risk of drug interactions. The particularity of these new drugs is a priori a pharmacokinetic and pharmacodynamic profile more predictable involving no need for laboratory monitoring. In some clinical situations (risk of too high or too low exposure), a specific dose is proposed.

Cellular expression and biological activities of alternatively spliced forms of tissue factor pathway inhibitor.

PURPOSE OF REVIEW: Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits tissue factor-factor VIIa (TF-fVIIa) and factor Xa (fXa). Recent studies revealed distinct cellular expression patterns for TFPIα and TFPIß and spurred additional experiments to define unique functions for these alternatively spliced TFPI isoforms. RECENT FINDINGS: TFPIα is produced by endothelial cells, localizes to an intracellular granule, and is released following cellular stimulation with thrombin or heparin. TFPIα also is produced by megakaryocytes and released from activated platelets. Platelet TFPIα limits clot growth following vessel injury and alters bleeding in hemophilia, suggesting that its primary physiological role is modulation of clot development. TFPIß is made by endothelial cells, localizes to the endothelium surface, and is not in platelets. TFPIß is an effective inhibitor of TF-mediated cellular migration and may act to dampen the adverse effects of intravascular TF expressed during inflammation. SUMMARY: Knowledge of TFPI isoform expression and activity provides new insights into the biochemical regulation of TF-mediated thrombotic and inflammatory disease. Recent findings have therapeutic implications for use of recombinant TFPI to treat severe sepsis in community-acquired pneumonia or to achieve improved engraftment of hematopoietic stem cells, and for development of TFPI-blocking pharmaceuticals to treat hemophilia.

Alterations of fibrin network structure mediated by dermatan sulfate.

Dermatan sulfate (DS) is well-known for its anticoagulant activity through binding to heparin cofactor II (HCII) to enhance thrombin inhibition. It has also been reported that DS has a profibrinolytic effect. We have evaluated the effects of DS solutions (4-20 µg/mL) on the formation (by kinetic studies), structure (by electron microscopy and compaction assays) and lysis (with urokinase-type plasminogen activator) of plasma fibrin networks. The results showed that DS significantly prolonged the lag phase and decreased the fibrin formation rate and the optical density of the final networks versus control, in a concentration dependent way. DS-associated networks presented a minor network percentage compared with control, composed of lower number of fibers per field, which resulted significantly thinner and longer. Moreover, DS rendered gels more sensible to rupture by centrifugal force and more susceptible to lysis. When fibrin formation kinetic assays were performed with purified fibrinogen instead of plasma, in the absence of HCII, the optical density of final DS-associated networks was statistically lower than control. Therefore, a direct effect of DS on the thickness of fibers was observed. Since in all in vitro assays low DS concentrations were used, it could be postulated that the fibrin features described above are plausible to be found in in vivo thrombi and therefore, DS would contribute to the formation of less thrombogenic clots.

Endogenous anticoagulants.

Blood coagulation is a complex and highly coordinated process that is constantly altered and impacted by procoagulant and anticoagulant "players." It is vital that these components work in concert to maintain a balance to keep coagulation in check. Several important endogenous anticoagulants will be discussed in this review including tissue factor pathway inhibitor, antithrombin, protein C, and protein S in origin, structure, mechanism of action, effects of deficiency, and current knowledge in veterinary medicine.

Effect of antithrombin III on glycoprotein Ib/IX/V activation in human platelets: suppression of thromboxane A2 generation.

We have previously shown that ristocetin, an activator of glycoprotein Ib/IX/V, induces release of soluble CD40 (sCD40) ligand via thromboxane (TX) A(2) production from human platelets. In the present study, we investigated the effect of antithrombin-III (AT-III), an anticoagulant, on the ristocetin-induced glycoprotein Ib/IX/V activation in human platelets. AT-III inhibited ristocetin-stimulated platelet aggregation. The ristocetin-induced production of 11-dehydro-TXB(2), a stable metabolite of TXA(2), and the release of sCD40 ligand were suppressed by AT-III. AT-III also reduced the ristocetin-stimulated secretion of platelet-derived growth factor (PDGF)-AB. AT-III failed to affect U46619-, a TXA(2) receptor agonist, induced levels of p38 mitogen-activated protein kinase phosphorylation or sCD40 ligand release. AT-III reduced the binding of SZ2, a monoclonal antibody to the sulfated sequence in the α-chain of glycoprotein Ib, to the ristocetin-stimulated platelets. These results strongly suggest that AT-III reduced ristocetin-stimulated release of sCD40 ligand due to inhibiting TXA(2) production in human platelets.

Enhanced thrombin generation and depressed anticoagulant function in children with pneumonia.

AIMS: To clarify the status of the coagulation system in children with community-acquired pneumonia. METHODS: Coagulation activation markers (prothrombin fragment F1 + 2, thrombin-antithrombin complexes, D-dimer), the natural anticoagulants (antithrombin, protein C and S) and tissue factor were measured in 28 consecutive children with pneumonia on admission to the hospital. Patients were divided into those with either bacterial-type pneumonia (at least two of the following three criteria: plasma C-reactive protein (CRP) >80 mg/L, white blood cell count >15 × 10(9) /L and alveolar infiltrates on the chest radiograph) or viral-type pneumonia. RESULTS: The majority of the patients (79%) showed elevation of at least one of the three coagulation activation markers. Plasma CRP concentration correlated with F1 + 2 (R = 0.44, p < 0.05) and D-dimer (R = 0.71, p < 0.0001). Patients with bacterial-type pneumonia (n = 17) had higher D-dimer levels (p < 0.05) and lower levels of antithrombin (p = 0.005) and protein C (p = 0.08) than the patients with viral-type pneumonia. CONCLUSIONS: Children with community-acquired bacterial-type pneumonia show distinctive changes in their coagulation system. The finding of coagulation system activation and depressed function of natural anticoagulants in uncomplicated pneumonia helps to understand the rapid and unpredictable changes observed in the coagulation status in patients with more severe forms of disease.

Bacterial lipoprotein TLR2 agonists broadly modulate endothelial function and coagulation pathways in vitro and in vivo.

TLR2 activation induces cellular and organ inflammation and affects lung function. Because deranged endothelial function and coagulation pathways contribute to sepsis-induced organ failure, we studied the effects of bacterial lipoprotein TLR2 agonists, including peptidoglycan-associated lipoprotein, Pam3Cys, and murein lipoprotein, on endothelial function and coagulation pathways in vitro and in vivo. TLR2 agonist treatment induced diverse human endothelial cells to produce IL-6 and IL-8 and to express E-selectin on their surface, including HUVEC, human lung microvascular endothelial cells, and human coronary artery endothelial cells. Treatment of HUVEC with TLR2 agonists caused increased monolayer permeability and had multiple coagulation effects, including increased production of plasminogen activator inhibitor-1 (PAI-1) and tissue factor, as well as decreased production of tissue plasminogen activator and tissue factor pathway inhibitor. TLR2 agonist treatment also increased HUVEC expression of TLR2 itself. Peptidoglycan-associated lipoprotein induced IL-6 production by endothelial cells from wild-type mice but not from TLR2 knockout mice, indicating TLR2 specificity. Mice were challenged with TLR2 agonists, and lungs and plasmas were assessed for markers of leukocyte trafficking and coagulopathy. Wild-type mice, but not TLR2 mice, that were challenged i.v. with TLR2 agonists had increased lung levels of myeloperoxidase and mRNAs for E-selectin, P-selectin, and MCP-1, and they had increased plasma PAI-1 and E-selectin levels. Intratracheally administered TLR2 agonist caused increased lung fibrin levels. These studies show that TLR2 activation by bacterial lipoproteins broadly affects endothelial function and coagulation pathways, suggesting that TLR2 activation contributes in multiple ways to endothelial activation, coagulopathy, and vascular leakage in sepsis.

The link between vascular features and thrombosis.

The mechanisms of vascular control of thrombotic events remain unclear. The vasculature possesses essential anticoagulant factors that regulate coagulation. Because the endothelium-to-blood ratios are much higher in the microcirculation, it is likely that stasis contributes to thrombotic risk, due in large part to failure to rapidly access the microcirculation and to gain access to this highly anticoagulant environment. Inflammation can potentiate thrombosis in part through downregulation of the vascular anticoagulants, a process that appears to be exacerbated in aging, a well-known risk factor for thrombosis. Surgery and trauma, two major risk factors for thrombosis, result in the release of a variety of cellular components that trigger coagulation through separate mechanisms.

Temporal changes in expression of FoxA1 and Wnt7A in isolated adult human alveolar epithelial cells enhanced by heparin.

Pre- and postnatal developmental studies of the lung have provided compelling evidence demonstrating multiple factors that orchestrate alveolar epithelial cell differentiation. The extent to which reactivation of certain developmental pathways in the adult might influence the course of differentiation of alveolar type 2 cells (AT2) into AT1 cells is not known. In this study, we examined selected members of the forkhead (Fox) family of transcription factors and the Wnt (wingless) family of signaling proteins for expression during human alveolar cell differentiation in vitro and determined their potential responses to sulfated components of extracellular matrix (ECM), like those shed from cell surfaces or found in basement membrane and modeled by heparin. Isolated adult human AT2 cells cultured over a 9-day period were used to define the temporal profile of expression of targeted factors during spontaneous differentiation to AT1-like cells. FoxA1 protein was upregulated at early to intermediate time points, where it was strongly elevated by heparin. Gene expression of wnt7A increased dramatically beginning on day 3 and was enhanced even further on days 7 and 9 by heparin, whereas protein expression appeared at days 7 and 9. These temporal changes of expression suggest that sulfated ECMs may act to enhance the increase in FoxA1 at the critical juncture when AT2 cells commence the differentiation process to AT1 cells, in addition to enhancing the increase in wnt7A when the AT1 cell phenotype stabilizes. Collectively, these factors may act to modulate differentiation in the adult human pulmonary alveolus.

Direct expression of recombinant activated human protein C, a serine protease.

Human protein C, like other serine proteases, is normally secreted as an inactive zymogen. It is converted to its active form extracellularly by limited proteolysis with the thrombin-thrombomodulin complex. This activation results from the removal of a 12-residue activation peptide from the NH2 terminus of the heavy (COOH-terminal) chain. We report here a successful strategy for the activation of human protein C during post-translational cellular processing, resulting in the secretion of activated protein C from transfected mammalian cells. Deletion of the nucleotides encoding the activation peptide resulted in the expression of a protease with less than 5% of the expected activity. However, the replacement of the activation peptide with an 8-residue sequence (Pro-Arg-Pro-Ser-Arg-Lys-Arg-Arg) involved in the proteolytic processing of the human insulin receptor precursor resulted in the direct expression of fully activated protein C. The mutant protein was shown to be correctly processed by NH2-terminal sequence analysis. This strategy for successful expression of an activated form of protein C may apply to the expression of active forms of other proteases which are naturally expressed as zymogens.

Removal of the anticoagulant activities of the low molecular weight heparin fractions and fragments with flavobacterial heparinase.

Recently, the development of low molecular weight heparin fractions and fragments (LMHF) as potential antithrombotic agents has gained increased attention. However, the lack of antagonists to neutralize the anticoagulant effects of these drugs may seriously exclude them from possible uses in extracorporeal therapy. This is mainly because of the concern that the high dosage of the drugs employed in extracorporeal therapy could lead to serious bleeding risks. Our earlier work has demonstrated that immobilized heparinase can remove polydisperse heparin both in vitro and in vivo. To examine whether such a system may be used as a novel approach to neutralize the anticoagulant effects of LMHF, different LMHF were tested using heparinase. In vitro data showed that both the APTT and anti-FXa activities of the LMHF including Kabi 2165, PK 10169, Cy 216 and CY 222 were nearly completely eliminated by heparinase in less than 20 min. This study suggests that an immobilized heparinase system may be an useful element for the acceptance of the LMHF for their use in extracorporeal therapy.

Anticoagulant and antithrombotic properties of a gamma-carboxyglutamic acid-rich peptide derived from the light chain of blood coagulation factor X.

In this report, we describe the anticoagulant and antithrombotic properties of a peptide (residues 1-44) derived from the amino-terminus of the bovine Factor X light chain by limited proteolysis with chymotrypsin, and subsequently purified by QAE-Sephadex chromatography. The effect of Factor X gla-peptide on the activation of human 3H-Factors IX and X was studied using radiometric assays and purified coagulation factors. Factor VIIa-tissue factor catalyzed activation of Factors IX and X was half-maximally inhibited by Factor X gla-peptide at concentrations of 0.8 microM and 0.2 microM, respectively. Factor IXa-VIII catalyzed Factor X activation was half-maximally inhibited at a gla-peptide concentration of 0.5 microM. In addition, thrombin formation by platelets incubated with Factor Xa and prothrombin could be similarly blocked by gla-peptide. Studies with bovine aortic endothelial cells indicated that the Factor X gla-peptide blocked in parallel Factor X binding and activation on the cell surface. Decarboxylation of the peptide by acid heat treatment destroyed its anticoagulant activity. The in vivo anticoagulant potential of native gla-peptide was demonstrated by a rapid prolongation of the PT and APTT following intravenous infusion into a rabbit. In addition, gla-peptide prevented thrombus formation in response to Factors IXa and Xa, but not thrombin, in a Wessler venous stasis model.

Antihemostatic, antiinflammatory, and immunosuppressive properties of the saliva of a tick, Ixodes dammini.

Pilocarpine-induced saliva of the tick, Ixodes dammini, inhibited platelet aggregation triggered by ADP and collagen, as well as platelet-aggregation factor. In addition, we found apyrase activity (which degrades ATP and ADP to AMP and orthophosphate) and an anticoagulant. We showed the presence of prostaglandin E2 (PGE2) by bioassay and radioimmunoassay. This saliva inhibited interleukin 2 production by T cell hybridomas, an activity consistent with that of PGE2. A kininase was demonstrated, and this may counteract the algesia- and edema-promoting properties of PGE2. Together, these salivary components produce antihemostatic, antiinflammatory, and immunosuppressive effects that may facilitate feeding, as well as transmission of tick-borne pathogens.

[Biotoxic effects of aqueous humour-a unifying pathogenetic theory, based on certain hypothetic biotoxic factors in aqueus (author's transl)]. / Biotoxizität des Kammerwassers. Eine vereinheitlichende pathologische Theorie, begründet auf hypothetische biotoxische Kammerwasserfaktoren

A hypothesis is proposed attributing a number of biotoxic effects to aqueous humor (ABC equals Aqueous Biotoxic Complex). Under normal anatomical and physiological conditions aqueous humour is contained in the anterior and posterior chamber reservoir and the ABC effects remain silent because the boundaries of the aqueous humour reservoir are resistant to these biotoxic factors. These factors are biochemically undefined but fall in the category of collagenolysins and anti-vascular enzymes (Prostaglandins?). Outside the normal reservoir these biotoxic effects result in a specific pathology such as corneal edema, subconjunctival edema, lens fiber edema, macular edema, papilledema and chorioidal edema.