Anti-hemostatic Factors Research Articles

Coagulation disorders in liver cirrhosis - Diagnostics and management

Patients with liver cirrhosis often exhibit complex alterations in their hemostatic system that can be associated with both bleeding and thrombotic complications. While prophylactic correction of abnormal coagulation parameters should be avoided, an individualized approach is recommended prior to invasive procedures, whereby specific preventive measures to stabilize hemostasis should be based on the periprocedural bleeding risk. While the haemostatic system of patients with compensated cirrhosis is often in a rebalanced haemostatic state due to a parallel decline in both pro- and anti-haemostatic factors, a decompensation of liver cirrhosis can lead to destabilization of this fragile equilibrium. Since conventional coagulation tests do not adequately capture the complex changes in the hemostatic system in cirrhosis, functional analysis methods such as viscoelastic tests or thrombin generation assays can be used for evaluating the coagulation status. This review describes the underlying pathophysiological changes in the hemostatic system in liver cirrhosis, provides an overview of diagnostic methods and discusses therapeutic measures in case of bleeding and thrombotic complications.

Acquired bleeding disorders.

Acquired bleeding disorders can accompany hematological, neoplastic, autoimmune, cardiovascular or liver diseases, but can sometimes also arise spontaneously. They can manifest as single factor deficiencies or as complex hemostatic abnormalities. This review addresses (a) acquired hemophilia A, an autoimmune disorder characterized by inhibitory autoantibodies against coagulation factor VIII; (b) acquired von Willebrand syndrome in patients with cardiovascular disorders, where shear stress abnormalities result in destruction of von Willebrand factor; and (c) liver function disorders that comprise complex changes in pro- and anti-hemostatic factors, whose clinical implications are often difficult to predict. The article provides an overview on the pathophysiology, diagnostic tests and state-of-the-art treatment strategies.

Global hemostatic status in patients with acute‐on‐chronic liver failure and septics without underlying liver disease

Unlabelled Box AbstractBackgroundEven the sickest patients with chronic liver disease (CLD), such as those with acute‐on‐chronic liver failure (ACLF) remain in hemostatic balance due to a concomitant decline in pro‐ and antihemostatic factors. ObjectivesWe aimed to study whether the hemostatic status in ACLF is merely an exaggeration from the status in patients with compensated and acutely decompensated cirrhosis, or whether sepsis‐associated hemostatic changes contribute. MethodsWe performed extensive hemostatic profiling in 31 adult patients with ACLF, 20 patients with sepsis without underlying CLD, and 40 healthy controls. ResultsWe found similarly elevated plasma levels of the platelet adhesive protein von Willebrand factor (VWF) and decreased levels of the VWF‐regulating protease ADAMTS13 in both groups compared to healthy controls. In vivo markers of activation of coagulation (thrombin‐antithrombin III, D‐dimer) were similarly elevated in both groups compared to controls, but ex vivo thrombin‐generating capacity was similar between patients and controls, despite a much more profound international normalized ratio elevation in ACLF. Plasma fibrinogen levels were much higher in septics, which was accompanied by a decreased ex vivo clot permeability and an increase in ex vivo resistance to clot lysis. All hemostatic parameters were remarkably stable over the first 10 days after admission. ConclusionsWe have found hemostatic changes in ACLF to partially overlap with that of patients with sepsis, and evidence of preserved hemostatic capacity in both patient groups. The notable difference was a profound hyperfibrinogenemia, associated with a thrombogenic clot structure and a marked ex vivo resistance to fibrinolysis in patients with sepsis.

Acquired bleeding disorders.

Acquired bleeding disorders can accompany hematological, neoplastic, autoimmune, cardiovascular or liver diseases, but can sometimes also arise spontaneously. They can manifest as single factor deficiencies or as complex hemostatic abnormalities. This review addresses (a) acquired hemophilia A, an autoimmune disorder characterized by inhibitory autoantibodies against coagulation factor VIII; (b) acquired von Willebrand syndrome in patients with cardiovascular disorders, where shear stress abnormalities result in destruction of von Willebrand factor; and (c) liver function disorders that comprise complex changes in pro- and anti-hemostatic factors, whose clinical implications are often difficult to predict. The article provides an overview on the pathophysiology, diagnostic tests and state-of-the-art treatment strategies.

Physiological and immunological responses to Culicoides sonorensis blood-feeding: a murine model

BackgroundHematophagous Culicoides spp. biting midges are of great agricultural importance as livestock, equine, and wildlife pests and as vectors of the orbiviruses bluetongue, epizootic hemorrhagic disease and African horse sickness. To obtain a blood meal, midges deposit saliva containing allergens, proteases, and anti-hemostatic factors, into the dermis to facilitate feeding. Infected midges deposit virus along with the myriad of salivary proteins during feeding. The extreme efficiency with which midges are able to transmit orbiviruses is not clearly understood, as much is still unknown about the physiological trauma of the bite and immune responses to saliva deposited during feeding. Of particular interest are the first few hours and days after the bite; a critical time period for any midge-transmitted virus to quickly establish a localized infection and disseminate, while avoiding the hosts’ immune responses.ResultsA mouse-midge feeding model using colonized Culicoides sonorensis midges was used to characterize innate mammalian immune responses to blood-feeding. Histological analysis of skin, and cellular and cytokine profiles of draining lymph nodes show Culicoides midge feeding elicited a potent pro-inflammatory Th-mediated cellular response with significant mast cell activation, subcutaneous hematomas, hypodermal edema and dermal capillary vasodilation, and rapid infiltration of leukocytes to the bite sites. Mast cell degranulation, triggered by bite trauma and specifically by midge saliva, was key to physiological and immunological responses and the ability of midges to feed to repletion.ConclusionsMidge feeding causes physiological and immunological responses that would be highly favorable for rapid infection and systemic dissemination orbiviruses if delivered during blood-feeding. Recruitment of leukocytic cells to bitten skin brings susceptible cell populations in proximity of deposited virus within hours of feeding. Infected cells would drain to lymph nodes, which become hyperplastic in response to saliva, and result in robust viral replication in expanding cell populations and dissemination via the lymph system. Additionally, saliva-induced vasodilation and direct breaches in dermal capillaries by biting mouthparts exposes susceptible vascular endothelial cells, thereby providing immediate sites of virus replication and a dissemination route via the circulatory system. This research provides insights into the efficiency of Culicoides midges as orbivirus vectors.

Spot sign as a predictor of rebleeding after endoscopic surgery for intracerebral hemorrhage.

In patients with spontaneous intracerebral hemorrhage (sICH), postoperative recurrent hemorrhage (PRH) is one of the most severe complications after endoscopic evacuation of hematoma (EEH). However, no predictors of this complication have been identified. In the present study, the authors retrospectively investigated whether PRH can be preoperatively predicted by the presence of the spot sign on CT scans. In total, 143 patients with sICH were treated by EEH between June 2009 and March 2017, and 127 patients who underwent preoperative CT angiography were included in this study. Significant correlations of PRH with the patients' baseline, clinical, and radiographic characteristics, including the spot sign, were evaluated using multivariable logistic regression models. The incidence of and risk factors for PRH were assessed in 127 patients with available data. PRH occurred in 9 (7.1%) patients. Five (21.7%) cases of PRH were observed among 23 patients with the spot sign, whereas only 4 (3.8%) cases of PRH occurred among 104 patients without the spot sign. The spot sign was the only independent predictor of PRH (OR 5.81, 95% CI 1.26-26.88; p = 0.02). The following factors were not independently associated with PRH: age, hypertension, poor consciousness, antihemostatic factors (thrombocytopenia, coagulopathy, and use of antithrombotic drugs), the location and size of the sICH, other radiographic findings (black hole sign and blend sign), surgical duration and procedures, and early surgery. The spot sign is likely to be a strong predictor of PRH after EEH among patients with sICH. Complete and careful control of bleeding in the operative field should be ensured when surgically treating such patients. New surgical strategies and procedures might be needed to improve these patients' outcomes.

Ticks elicit variable fibrinogenolytic activities upon feeding on hosts with different immune backgrounds

Ticks secrete several anti-hemostatic factors in their saliva to suppress the host innate and acquired immune defenses against infestations. Using Ixodes scapularis ticks and age-matched mice purchased from two independent commercial vendors with two different immune backgrounds as a model, we show that ticks fed on immunodeficient animals demonstrate decreased fibrinogenolytic activity in comparison to ticks fed on immunocompetent animals. Reduced levels of D-dimer (fibrin degradation product) were evident in ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Increased engorgement weights were noted for ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Furthermore, the LC-MS/MS and quantitative real-time-PCR analysis followed by inhibitor and antibody-blocking assays revealed that the arthropod HSP70-like molecule contributes to differential fibrinogenolysis during tick feeding. Collectively, these results not only indicate that ticks elicit variable fibrinogenolysis upon feeding on hosts with different immune backgrounds but also provide insights for the novel role of arthropod HSP70-like molecule in fibrinogenolysis during blood feeding.

Rebalanced Hemostasis in Patients with Acute Liver Failure.

Patients with acute liver failure (ALF) have substantial alterations in their hemostatic system. Since an international normalized ratio of ≥ 1.5 is part of the definition of the syndrome, it has long been believed that patients with ALF had a hemostasis-related bleeding tendency. Recent data, however, show that spontaneous bleeding in ALF is rare. In addition, thrombotic complications may be more common than spontaneous bleeding complications. Laboratory studies have suggested that patients with ALF may be in hemostatic balance as a result of a commensurate decline in pro- and anti-hemostatic factors. The unstable nature of the hemostatic balance in ALF may explain the occurrence of both bleeding and thrombotic complications. The hemostatic profile of patients with ALF includes hypercoagulable features, including von Willebrand factor/a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 unbalance, elevated levels of highly procoagulant, platelet-derived microparticles, and a profound hypofibrinolytic status. These hypercoagulable features may contribute to systemic thrombotic complications, but may also drive intrahepatic clot formation. Studies in experimental animal models of ALF have demonstrated that intrahepatic clot formation contributes to disease progression. The clinical consequences of these new insights in the hemostatic system of patients with ALF will be discussed in this review.

Characterization of thrombin inhibitory mechanism of rAaTI, a Kazal-type inhibitor from Aedes aegypti with anticoagulant activity

Saliva of blood-sucking arthropods contains a complex mixture of anti-haemostatic, anti-inflammatory and immune-modulator compounds. Among anti-haemostatic factors, there are anticoagulants, vasodilators and platelet aggregation inhibitors. Previous analyses of the sialotranscriptome of Aedes aegypti showed the potential presence of a Kazal-type serine protease inhibitor in the female salivary glands, carcass and also in the whole male, which inhibitor we named AaTI (A. aegypti thrombin inhibitor). Recently, we expressed and characterized rAaTI as a trypsin inhibitor, and its anticoagulant activity [1]. In this work we characterized the thrombin inhibition mechanism of rAaTI. Recombinant AaTI was able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. In contrast, AaTIΔ (rAaTI truncated form) and C-terminal AaTI acidic tail prolong only thrombin time. In the competition assay, rAaTI, AaTIΔ or C-terminal AaTI acidic tail–thrombin interactions seem to be affected by heparin but not by hirudin, suggesting that rAaTI binds to thrombin exosite 2. Finally, the thrombin inhibition assay of rAaTI showed an uncompetitive inhibition mechanism. In conclusion, rAaTI can probably inhibit thrombin by interacting with thrombin exosite 2, and the interaction is not mediated by the AaTI C-terminal region, since the truncated AaTIΔ form also prolongs thrombin time.

Immunization with Adenoviral-expressed salivary gland proteins (SALPs) decreases spirochete load in a murine model of Lyme borreliosis (52.2)

Abstract It is known that tick saliva contains a vast array of proteins that function as anti-haemostatic, anti-inflammatory, and immunomodulatory factors when delivered to the vertebrate host. Previous studies have also demonstrated that prior exposure to tick saliva induced specific immunity to tick infestation, as well as affording protection against tick-transmitted Borrelia burgdorferi infection in the mammalian host. In our current studies we explored a vaccination technique to modulate tick feeding, using an adenovirus (Ad) expression system to deliver four salivary proteins (previously shown to modulate host immunity) derived from Ixodes scapularis; Salp15, Salp25A, Salp25D, and ISAC. C3H/HeJ mice were immunized individually with Ad-Salp15 and Ad-ISAC constructs, as well as all four constructs simultaneously. Immunized mice were then challenged with B. burgdorferi-infected I. scapularis ticks. Utilizing quantitative PCR, spirochete burden in target organs like heart and bladder was reduced by 55% compared to animals immunized with an empty Ad-cassette. The generation of anti-salivary protein antibodies in a murine model was also confirmed. Our results indicate that adenovirus expressed tick salivary proteins can stimulate dendritic cell expression of tick salivary proteins, as well as tick-specific humoral and cellular immune responses in mice, affording at least partial protection against subsequent tick-transmitted B. burgdorferi infection.

Black fly salivary gland extract inhibits proliferation and induces apoptosis in murine splenocytes

Black flies are known to be vectors of pathogens including Onchocerca volvulus, which causes human onchocerciasis, and Vesicular Stomatitis Virus. Their salivary secretion has been shown to contain a complex cocktail of anti-haemostatic factors and immunomodulatory activities, which may contribute to efficient transmission of the pathogens. Black fly salivary gland extract (SGE) inhibits mitogen-stimulated mouse splenocyte proliferation, including proliferation of both CD4(+) and CD8(+) T cells. The factor responsible for the inhibition was determined to be a protein (or protein complex) of a size larger than 50 kDa. Moreover, exposure to SGE results in activation of caspase 3 and characteristic morphological changes in CD4(+) and CD8(+) T cells, suggesting that induction of apoptosis could, at least in part, be responsible for this inhibition.

Characterization of anti-hemostatic factors in the argasid, Argas monolakensis: Implications for the evolution of blood-feeding in the soft tick family

To date, the only anti-hemostatic factors characterized for softs ticks are for Ornithodoros moubata and Ornithodoros savignyi, ticks that feeds mainly on mammals. This includes thrombin (ornithodorin and savignin), fXa (TAP and fXaI) and platelet aggregation (disagegin and savignygrin) inhibitors that belong to the BPTI-Kunitz protein family. This raises the question on how well anti-hemostatic factors will be conserved in other soft tick genera that feeds on other vertebrates such as birds. We characterized the anti-hemostatic factors from Argas monolakensis, a soft tick that feeds mainly on Californian gulls. The main anti-clotting factor (monobin) is an ortholog of ornithodorin and savignin and shows similar slow tight-binding kinetics. The main anti-platelet activities are apyrase and fibrinogen receptor antagonists (monogrins). The monogrins are orthologs of disagregin and savignygrin and like savignygrin presents the RGD integrin-recognition motif on the BPTI substrate-binding presenting loop. This implies that the anti-hemostatic factors evolved in the ancestral soft tick lineage and has been maintained in soft tick species from two distinct genera with different host preferences. The Argas derived anti-hemostatic factors bind to mammalian targets with affinities similar to that observed for their orthologs in the Ornithodoros genus. This cross-reactivity could have facilitated the switching of soft ticks from avian to mammalian hosts and can explain in part the ability of Argas ticks, to feed on humans, thereby remaining a possible health risk.

A reassessment of argasid tick salivary gland ultrastructure from an immuno-cytochemical perspective

Previous morphological and histochemical studies of argasid tick salivary glands indicated that they were less complex than ixodid salivary glands, with only three granular cell types. The present study shows that there exist at least four different granular cell types in the salivary glands of the argasid tick Ornithodoros savignyi, based on immuno-localization of the anti-hemostatic factors, apyrase and savignygrin. Both anti-hemostatic factors were localized to dense core granule type 'a' and to granule type 'b', that shares a similar homogenous morphology with non-labeled granule type 'd'. Furthermore, the major tick salivary gland proteins (TSGPs), previously implicated in granule biogenesis, were localized to all the granular cell types. This indicates that granular cell types with different morphologies can express the same proteins, while cell types that show similar morphologies may not express the same proteins. Argasid tick salivary glands seem to be more complex than previously thought and might not be amenable to morphological classification alone. Alternative classification methodologies that rely on physical expression patterns of the salivary gland proteome might be more reliable as markers for a specific granular cell type.

Lysophosphatidylcholine Acts as an Anti-hemostatic Molecule in the Saliva of the Blood-sucking Bug Rhodnius prolixus

Blood-sucking arthropods possess a variety of anti-hemostatic factors in their salivary glands to maintain blood fluidity during feeding. In this work we demonstrate the anti-hemostatic properties of lysophosphatidylcholine (lysoPC) isolated from the salivary glands of Rhodnius prolixus. First, we examined salivary glands of fourth and fifth instar nymphs for their phospholipid composition. The lumen displayed an accumulation of its phospholipid content, mainly phosphatidylcholine and lysoPC, with a 6-fold increase for the latter. To determine the presence of phospholipids in the saliva, fourth instar nymphs were fed with a32P-enriched blood meal. After 28 days their saliva was collected and subjected to lipid extraction, thin-layer chromatography, and autoradiography. The results showed the presence in the saliva of the same phospholipids present in the lumen. We then examined possible biological roles of these phospholipids when compared with other known effects of lysoPC. The luminal lipid extract and purified lysoPC from the lumen and saliva were tested for inhibition of washed rabbit platelets' aggregation induced by alpha-thrombin and platelet-activating factor. Both the luminal lipid extract and salivary lysoPC showed an increasing inhibition of aggregation, which correlated with the response of the platelets to standard lysoPC (up to 13 microg/ml). Next, salivary lysoPC was incubated with porcine arterial endothelial cells for 24 h. After incubation, culture medium was assayed for nitric oxide and showed increased nitric oxide production, similar to control cells exposed to standard lysoPC (up to 20 microg/ml). Together these data demonstrate the presence of lysoPC in the saliva of Rhodnius prolixus and its potential anti-hemostatic activities.

Thrombostasin: purification, molecular cloning and expression of a novel anti-thrombin protein from horn fly saliva

Thrombostasin (TS), a novel protein found in the saliva of Haematobia irritans (horn fly), was purified by high-performance liquid chromatography from the saliva of field-collected insects. This protein, which inhibits thrombin, accounts for anti-clotting activity in horn fly saliva [J. Med. Entomol. 37 (2000) 416] and is the first purified anti-hemostatic factor described from the Stomoxyinae, a large group of blood-feeding insects that are major pests of livestock world-wide. The purified TS had an apparent molecular weight of 16.7 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and revealed two isoelectric groups with isoelectric points (p Is) of approximately 4.6 and 4.8. Mass spectroscopy analysis, however, resulted in at least three major isoforms that range in mass from 9213 to 9274 Da. A 243-bp coding sequence was obtained from cDNA by using a degenerate primer deduced from the N-terminal sequence of the purified TS. The conceptual translation of the 243-bp sequence showed that the 81-amino-acid peptide, whose first 30 amino acids match those of the N-terminal sequence, had a predicted mass of 9213 Da with p I 4.14. A full-length TS cDNA was generated by rapid amplification of cDNA ends of the 5′ and sequential polymerase chain reaction (PCR) amplification. It contained a 5′-end 12-bp segment preceding the putative ATG start codon, followed by a 54-bp sequence corresponding to a secretory signal and an additional 228-bp coding sequence preceding residues revealed by N-terminal sequencing of purified TS. The fidelity of the PCR-generated TS sequence was confirmed in genomic DNA and by biological activity of recombinant TS produced in a baculovirus expression system. Database comparisons revealed no homology between TS and other known molecules. Because of the paucity of other anti-hemostatic factors in horn fly saliva, TS may play a critical role in maintenance of the ectoparasitic lifestyle of horn flies.

Evolution of anti-hemostatic factors from soft ticks of the Ornithodoros spp. (Acari: Argasidae)

Evolution of anti-hemostatic factors from soft ticks of the <i>Ornithodoros</i> spp. (Acari: Argasidae)

Blood-feeding strategy of Haematobia irritans (Diptera: Muscidae).

The economic impact on livestock production by Haematobia irritans (L.) is estimated to approach $1 billion per year in North America. However, there is little information regarding the blood-feeding strategy used by these insects. Information presented here shows that horn fly saliva interferes with the normal coagulation response as measured by the recalcification time assay. The relative anticoagulant activity on a per-gland basis was more than or equal to that reported for Simulium vittatum Zetterstedt, a common hematophagous black fly that also feeds on cattle. However, unlike S. vittatum, H. irritans salivary factors do not inhibit platelet aggregation using apyrase and have no detectable vasodilative activity. In this regard, the horn fly is strikingly different from blood-feeding species in the lower Diptera and shows a much more limited repertoire of antihemostatic factors.

Apyrase activity and platelet aggregation inhibitors in the tick Ornithodoros savignyi (Acari: Argasidae).

Ticks are ectoparasites that cause considerable damage to their hosts while feeding. The feeding process is facilitated by anti-haemostatic factors present in the tick saliva. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is a platelet aggregation inhibitor found in most haematophagous organisms studied. The present study describes the identification and characterization of such an activity in the tick Ornithodoros savignyi. The enzyme conformed to many properties common to apyrases. These included a low substrate specificity, dependence on bivalent metal ions for activity and insensitivity to the classical ATPase inhibitors. Heat denaturation studies, pH optima and similar effects of inhibitors on the enzyme's ATP and ADP hydrolysing activitives supported its classification as an apyrase. Salivary gland extracts inhibited the platelet aggregation induced by ADP, collagen and thrombin and disaggregated aggregated platelets. The results suggest the presence of two or more anti-platelet factors present in the salivary glands of this tick species.

The role of the leech in medical therapeutics

Leeching is considered by many to be a discredited medical relic of the past. This view is not justified, since leeches still play an important part in modern medicine, as in microsurgery and in the treatment of patients with post-phlebitic syndrome. Hirudin, the potent thrombin inhibitor of leech saliva, has been cloned and is used in the treatment of cardiological and hematological disorders. In our search for other antihemostatic factors in Hirudo medicinalis saliva, we found inhibitors of platelet aggregation induced by thrombin, collagen, adenosine 5′-diphosphate, epinephrine, platelet-activating factor and arachidonic acid. We purified apyrase (adenosine 5′-dpphosphate diphosphohydrolase), which is a non-specific inhibitor of platelet aggregation by virtue of its action on adenosine 5′-diphosphate. We isolated and characterized the platelet-activating factor antagonist and also identified and recovered an inhibitor of coagulation factor Xa from leech saliva. This report summarizes our findings and those of other investigators, as well as the experience of one of us (A.E.) in leech therapy.

Tick salivary gland physiology.

The multifunctional, morphologically complex salivary glands are essential to the biological success of ticks and are intricately involved in the transmission of pathogens. They are innervated, and there is convincing evidence that dopamine is a neurotransmitter at the neuroeffector junction controlling fluid secretion. As feeding progresses, the rate of salivary fluid secretion increases greatly, enabling the ixodid tick to concentrate the bloodmeal by returning excess water and ions to the host. Saliva in feeding ticks is rich in bioactive components and exhibits a range of pharmacological properties. Factors identified in saliva or salivary glands include cement to help anchor the mouthparts to the host, various enzymes and inhibitors, histamine agonists and antagonists, prostaglandins, antihemostatic factors, and immuno-modulating factors. A secretion from the salivary glands allows ticks to absorb water from the air during the lengthy periods off their hosts. The physiology of this remarkable organ provides a striking example of strategies that have evolved to meet the challenge of a unique parasitic life style.