Newly Identified HNP‐F from Human Neutrophil Peptide‐1 Promotes Hemostasis

Active hemostatic agents can play a crucial role in saving patients’ lives during surgery. Active hemostats have several advantages including utilization of natural blood coagulation and biocompatibility. Among them, although human neutrophil peptide‐1 (HNP‐1) has been previously reported with the hemostatic mechanism, which part of HNP‐1 facilitates the hemostatic activity is not known. Here, a partial peptide (HNP‐F) promoting hemostasis, originating from HNP‐1, has been newly identified by the blood coagulation ability test. HNP‐F shows the best hemostatic effect between the anterior half and posterior half of peptides. Moreover, microscopic images show platelet aggregation and an increase in the concentration of platelet factor 4, and the scanning electron microscope image of platelets support platelet activation by HNP‐F. Thromboelastography indicates decreased clotting time and increased physical properties of blood clotting. Mouse liver experiments demonstrate improved hemostatic effect by treatment of peptide solution. Cell viability and hemolysis assays confirm the HNP‐F's biosafety. It is hypothesized that the surface charge and structure of HNP‐F could be favorable to interact with fibrinogen or thrombospondin‐1. Collectively, because HNP‐F as an active peptide hemostat has many advantages, it could be expected to become a potent hemostatic biomaterial, additive or pharmaceutical candidate for various hemostatic applications.     

Evaluation of vasopressin mediated effects on hemostatic mechanisms: relationship with aquaporins and caveolin proteins

With a view to evaluate the role of AQP-1 and caveolin proteins in the hemostatic actions of vasopressin, hemostasis was evaluated by bleeding and clotting time respectively.Groups of mice and guinea pigs were treated with arginine vasopressin (AVP) and 1-deamino-8D-AVP (DDAVP) to evaluate their effects on the hemostasis. DDAVP and AVP were able to appreciably reduce the bleeding and clotting time after sodium thiopentone, but not effectively after TEA treatment. Animal groups were pretreated with aquaporin-1 (AQP-1) blockers or water deprived to enhance the expression of AQP-1 water channels. Another group of animals were treated with caveolin protein modulators, cholera toxin (CTX) and the effect of vasopressin analogues evaluated. The results suggest that AQP-1 water channels and caveolin proteins contribute to modulate the hemostatic mechanisms of vasopressin.     

GNPs-CS/KGM as Hemostatic First Aid Wound Dressing with Antibiotic Effect: In Vitro and In Vivo Study

Ideal wound dressing materials should create a good healing environment, with immediate hemostatic effects and antimicrobial activity. In this study, chitosan/konjac glucomannan (CS/KGM) films embedded with gentamicin-loaded poly(dex-GMA/AAc) nanoparticles (giving GNP-CS/KGM films) were prepared as novel wound dressings. The results revealed that the modified CS/KGM films could be used as effective wound dressings and had significant hemostatic effects. With their microporous structure, the films could effectively absorb water from blood and trap blood cells. The gentamicinloaded poly(dex-GMA/AAc) nanoparticles (GNPs) also further promoted blood clotting, with their favorable water uptake capacity. Thus, the GNP-CS/KGM films had wound healing and synergistic effects that helped to stop bleeding from injuries, and also showed good antibiotic abilities by addition of gentamicin to the NPs. These GNPCS/KGM films can be considered as promising novel biodegradable and biocompatible wound dressings with hemostatic capabilities and antibiotic effects for treatment of external bleeding injuries.     

Dental extraction in congenital hemorrhagic patients

125 dental extractions were performed in patients with hemophilia A, B, von Willebrand disease and rare coagulopathies. The missing factor was brought to a level of 15-40% according to the degree of surgical trauma. In the majority of cases a single pre-extraction administration of concentrate was sufficient. Antifibrinolytics were administered to the patients (tranexamic acid, 60 mg/kg/day) for 8-10 days. After tooth extraction, a fibrin sponge was positioned using anti-traumatic cross suture. In 90% of cases there was no bleeding, in the others modest easily controllable hemorrhagic episodes. Our results show that it is sufficient in hemophilia A and B to use low doses of antihemophilic concentrate (10-20 U/kg) and frequently a single pre-extraction administration. In rare coagulopathies the hemostatic procedure and dental management have also to be similar to those used in hemophilia A and B.     

Possible origin of amniotic fluid constituents affecting thromboplastic activity

The clotting-accelerating activity of amniotic fluid (AF) has been known for long, but a clinical importance has only recently been attributed to this phenomenon. In order to obtain some informations on the origin of AF component(s) responsible for this action, the effects on the coagulation process of extracts prepared from the placenta, fetal membranes and fetal excretes were investigated. Normal saline extracts from the placenta, the amniotic and chorionic membranes, and those from the mucus aspirated from the upper respiratory tract of the fetuses were shown to accelerate, whereas meconium extract and untreated fetal urine were found to inhibit blood coagulation. Results presented in this paper indicate that AF components affecting the thromboplastic system in one way or other may be of both fetal and extrafetal gestational tissue origin, and that the quality of the actual effect exerted on the clotting system by AF depends on the relative amount of AF constituents oppositely affecting the hemostatic system.     

Influence of temperature on blood coagulation in vitro (author's transl)]

The influence of different temperatures between 13 degrees C and 45 degrees C on coagulation factors in vitro was studied by measuring clotting time with the recalcification time, partial thromboplastin time (PTT), and thromboplastin time test. In all three tests the shortest clotting times were measured at a temperature of 40 degrees C. The relation between temperature and clotting time was similar in fresh plasma and in plasma which had been stored at a temperature of --20 degrees C before examination. However, in all tests stored plasma showed shorter coagulation times. Prolongation of coagulation time at 45 degrees C is caused by irreversible reduction of coagulation activity in the plasma. At the same time thromboplastin- and PTT-reagent are imparied in their coagulation acitvity by a temperature of 45 decrees C. In comparison to plasma obtained from healthy persons plasma from patients with hemophilia A or B or with v. Willebrand's disease reacted more sensitive to changes in temperature in the PTT test. The coagulation defect was definitely more pronounced at 27 degrees and 17 degrees C than at 37 degrees C. It was not possible to differentiate these three coagulopathies with the PTT test at 27 degrees and 17 degrees C.     

Coagulation, hemostasis, and plasma expanders:a quarter century enigma.

Despite more than 2 decades of research, the explanation of the long-known hemostatic failure consequent to the use of some natural and synthetic macromolecular agents as plasma substitutes remains obscure. Conventional clotting parameters are not significantly affected in vivo or in vitro. Dextran, hydroxyethyl starch, and many other colloid macromolecules precipitate Factors I and VIII, fibrin monomer, and perhaps v. W. (von Willebrand) factor(s) from plasma, rendering at least the first three insoluble, in relation to the molecule size and concentration of the colloid, and for dextran, its intrinsic viscosity. The precipitate, rich in Factors VIII and I, redissolves on warming, and reprecipitates on cooling, behaving as a cryo-Factor I. In composition it closely resembles the cryoprecipitate obtained by slow-thawing of plasma. Both clot faster with thrombin than the parent plasma. The amount precipitated from plasma by dextran or hydroxyethyl starch varies very widely from individual to individual. Cryo- of dextran-precipitable material can be obtained by interacting purified Factor I with a miniscule amount of thrombin. Dextran, hydroxyethyl starch, polyvinyl pyrrolidone, some forms of gelatin, and several polyamino acids accelerate thrombin clotting of normal plasma, several dysfibrinogenemic plasmas, or Factor I. Albumin, hemoglobin, some modified gelatins do not. Poor platelet thromboplastic function appears some hours after dextran infusion, associated with morphologic capillary abnormalities that strikingly resemble those in v. W. disease. We postulate that the hemostatic defect associated with the use of plasma substitutes is a form of induced v. W. disease or disseminated intravascular clotting, ensuing from precipitation and removal of v. W. factor(s), Factors VIII and I, microcirculatory abnormality, and platelet malfunction. The latter two supervene some time after administration of dextran. It reported antithrombotic activity is perhaps referable to the same action.     

Simple shifts in redox/thiol balance that perturb blood coagulation.

The biological chemistry that underlies and regulates the blood coagulation cascade is not fully understood. To begin to understand this, we performed clotting assays under various redox conditions. By varying the amount of oxidant and/or antioxidant in these assays, we observed that both the intrinsic/tenase complex and the extrinsic pathways were susceptible to shifts in the thiol/redox balance. We established a dichotomy where blood clotting via the intrinsic pathway was sensitive to oxidation whereas the tissue factor or extrinsic pathway was more sensitive to reduction. These differential inhibitory effects present a conceptual mechanism for selective modulation of the activities of clotting factors specific for the respective pathways. These data also suggest that blood clotting may be influenced by unidentified redox or thiol equilibria.     

Biophysical mechanisms of contact activation of blood-plasma clotting

This review considers the biochemical and biophysical mechanisms that trigger blood clotting upon contact of blood with an alien surface and leads via a cascade of enzymatic reactions to fibrin polymerization and the formation of a blood plasma clot, which permeates a primary platelet aggregate to produce a dense hemostatic clot. In spite of the substantial number of experimental and theoretical studies on the subject, there is still no consistent opinion as to what processes occur as the blood plasma contacts a surface. This review discusses the role that plasma protein factor XII and various surfaces play in triggering the contact pathway in vivo and in vitro. Current views of the molecular events that underlie the process are described.     

Hemostatic changes induced by exercise during oral contraceptive use

Exercise-induced changes in hemostatic measurements were studied in 25 women. Twelve of the subjects were not using oral contraceptives and the remainder were using Demulen (ethynodiol diacetate (1 mg) and ethinyl estradiol (0.05 mg)). Exercise on a treadmill induced similar changes in both groups, but during the use of Demulen the levels of fibrinogen and plasminogen were higher, antithrombin level was lower, and the recalcified clotting and dilute whole blood lysis times were shorter in group 2 than in the corresponding samples obtained from the nonpill users.     

A novel swine model for evaluation of potential intravascular hemostatic agents

Because uncontrolled hemorrhage is a leading cause of battlefield mortality, finding an intravenous treatment that could assist endogenous clotting mechanisms is a major mission for military researchers. Evaluation of potential intravenous hemostatic agents requires both in vitro and in vivo tests. For in vivo evaluation, we have developed a novel swine model in which 1) bleeding times (BT) and coagulation function could be ascertained after multiple doses of hemostatic drug administration and 2) a subsequent exsanguinating injury could be performed in the same animal, yielding screening information regarding the effects of drug pretreatment on blood loss and survival. Transection of small mesenteric arteries and veins allowed for multiple and reproducible BT measures that correlated with coagulation function. Subsequent excision of defined areas of the liver produced bleeding predominantly from small vessels (diameter, less than 2 mm) and parenchyma while resulting in 62% mortality without the use of either heparinization or aggressive fluid infusion. This swine model allows for multiple, repeatable BT measures in the same animal in experiments already involving laparotomy. Such a model is well suited for terminal studies to test effects of multiple doses of the same drug or multiple drugs on BT and allows for multiple, easily visualized measures that permit enhanced repeatability. The liver injury provides for numerous small vessel lesions that could be amenable to closure by coagulation. Therefore, drugs or mechanisms that enhance coagulation and concomitantly decrease blood loss and increase survival time may be accurately evaluated in this new model.     

A computational thrombus formation model: application to an idealized two-dimensional aneurysm treated with bare metal coils

Cardiovascular implantable devices alter the biofluid dynamics and biochemistry of the blood in which they are placed. These perturbations can lead to thrombus formation which may or may not be desired, depending on the application. In this work, a computational model is developed that couples biofluid dynamics and biochemistry to predict the clotting response of blood to such devices. The model consists of 28 advection–diffusion–reaction partial differential equations to track proteins in the blood involved in clotting and utilizes boundary flux terms to model the initiation of the intrinsic clotting pathway at thrombogenic device surfaces. We use this model to simulate the transient clot growth within a 2D idealized bifurcation aneurysm filled with various distributions of bare metal coils with similar packing densities. The clot model predicts initial clot formation to occur in areas along coil surfaces where flow is minimal and where time-averaged shear rates are the smallest. Among the six coil-filled aneurysm cases simulated, maximum thrombus occlusion ranged between 80.8 and 92.2% of the post-treatment aneurysm volume and was achieved 325–450 s after treatment. With further refinement and validation, the computational clotting model will be a valuable engineering tool for evaluating and comparing the relative performance of cardiovascular implantable devices.     

Milk Clotting Enzyme from Microorganisms

Out of some 800 strains of microorganisms, a potent fungus for milk clotting enzyme was isolated from soil during the course of screening tests and was identified as one of strains of Mucor pusillus Lindt. Satisfactory results were obtained in cheese making experiments with this enzyme which could be produced effectively by solid culture on wheat bran at 30°C for about 70 hrs.

The balance between milk clotting activity and proteolytic activity of this enzyme resembled very much to that of rennet.

Microbial rennet from Mucor pusillus F-27 was obtained with high productivity by solid culture followed by water extraction. The enzyme could be precipitated by salting out with ammonium sulfate and also by mixing with various water-miscible organic solvents such as ethanol, methanol or acetone.

This enzyme is one of acid proteases having its optimal pH for milk casein digestion around 3.5. The ratio of milk clotting activity to proteolytic activity of this enzyme resembled that of calf rennet than those of other proteases of fungal origin. This was more heat stable and more resistant against pH changes than animal rennet. Apparent activity of milk clotting was more affected by Ca ion concentration in milk than that of calf rennet.

The liberation of 12% TCA soluble nitrogen from casein fraction was a little less specific than that of calf rennet. The optimal temperature for milk clotting lay around 56°C.

Electrophoretic patterns of α-peak of casein treated with this enzyme showed the weak proteolysis which resembled that with rennet.     

No effects without causes: the Iron Dysregulation and Dormant Microbes hypothesis for chronic,inflammatory diseases

Since the successful conquest of many acute, communicable (infectious) diseases through the use of vaccines and antibiotics, the currently most prevalent diseases are chronic and progressive in nature, and are all accompanied by inflammation. These diseases include neurodegenerative (e.g. Alzheimer's, Parkinson's), vascular (e.g. atherosclerosis, pre‐eclampsia, type 2 diabetes) and autoimmune (e.g. rheumatoid arthritis and multiple sclerosis) diseases that may appear to have little in common. In fact they all share significant features, in particular chronic inflammation and its attendant inflammatory cytokines. Such effects do not happen without underlying and initially ‘external’ causes, and it is of interest to seek these causes. Taking a systems approach, we argue that these causes include (i) stress‐induced iron dysregulation, and (ii) its ability to awaken dormant, non‐replicating microbes with which the host has become infected. Other external causes may be dietary. Such microbes are capable of shedding small, but functionally significant amounts of highly inflammagenic molecules such as lipopolysaccharide and lipoteichoic acid. Sequelae include significant coagulopathies, not least the recently discovered amyloidogenic clotting of blood, leading to cell death and the release of further inflammagens. The extensive evidence discussed here implies, as was found with ulcers, that almost all chronic, infectious diseases do in fact harbour a microbial component. What differs is simply the microbes and the anatomical location from and at which they exert damage. This analysis offers novel avenues for diagnosis and treatment.     

Decreased sensitivity of human platelets to PGI2 during long-term intraarterial prostacyclin infusion in patients with peripheral vascular disease — A rebound phenomenon?

During successful treatment of peripheral vascular disease with synthetic prostacyclin no alteration in platelet function was reported (1).In 8 patients infused with synthetic prostacyclin continuously for 7 days intraarterially, the platelet function was monitored. Special attention was drawn to the platelet sensitivity in vitro for PGI₂, which is discussed as an important factor maintaining the hemostatic balance.In all the patients with peripheral vascular disease between 24 and 48 hours after the beginning of the infusion a sudden decrease in platelet sensitivity accompanied by an increase in platelet count could be seen.These dramatic alterations representing probably a rebound phenomenon occurring during long-term PGI₂-treatment might be an explanation for a non-beneficial effect of the treatment and in some cases a limiting factor for the continuation of the infusion itself. It is not clear, if this rebound phenomenon is due to a stimulation of an endogenous inhibitor, lowering the synthesis of a naturally occurring substance acting against this inhibitor or tachyphylaxia.     

Microcirculation and oxidative stress

The microcirculation is a complex and integrated system, transporting oxygen and nutrients to the cells. The key component of this system is the endothelium, contributing to the local balance between pro and anti-inflammatory mediators, hemostatic balance, as well as vascular permeability and cell proliferation. A constant shear stress maintains vascular endothelium homeostasis while perturbed shear stress leads to changes in secretion of vasodilator and vasoconstrictor agents. Increased oxidative stress is a major pathogenetic mechanism of endothelial dysfunction by decreasing NO bioavailability, promoting inflammation and participating in activation of intracellular signals cascade, so influencing ion channels activation, signal transduction pathways, cytoskeleton remodelling, intercellular communication and ultimately gene expression. Targeting the microvascular inflammation and oxidative stress is a fascinating approach for novel therapies in order to decrease morbidity and mortality of chronic and acute diseases.     

Functional Variability of Snake Venom Metalloproteinases: Adaptive Advantages in Targeting Different Prey and Implications for Human Envenomation

Snake venom metalloproteinases (SVMPs) are major components in most viperid venoms that induce disturbances in the hemostatic system and tissues of animals envenomated by snakes. These disturbances are involved in human pathology of snake bites and appear to be essential for the capture and digestion of snake''s prey and avoidance of predators. SVMPs are a versatile family of venom toxins acting on different hemostatic targets which are present in venoms in distinct structural forms. However, the reason why a large number of different SVMPs are expressed in some venoms is still unclear. In this study, we evaluated the interference of five isolated SVMPs in blood coagulation of humans, birds and small rodents. P-III class SVMPs (fractions Ic, IIb and IIc) possess gelatinolytic and hemorrhagic activities, and, of these, two also show fibrinolytic activity. P-I class SVMPs (fractions IVa and IVb) are only fibrinolytic. P-III class SVMPs reduced clotting time of human plasma. Fraction IIc was characterized as prothrombin activator and fraction Ic as factor X activator. In the absence of Ca²⁺, a firm clot was observed in chicken blood samples with fractions Ic, IIb and partially with fraction IIc. In contrast, without Ca²⁺, only fraction IIc was able to induce a firm clot in rat blood. In conclusion, functionally distinct forms of SVMPs were found in B. neuwiedi venom that affect distinct mechanisms in the coagulation system of humans, birds and small rodents. Distinct SVMPs appear to be more specialized to rat or chicken blood, strengthening the current hypothesis that toxin diversity enhances the possibilities of the snakes for hunting different prey or evading different predators. This functional diversity also impacts the complexity of human envenoming since different hemostatic mechanisms will be targeted by SVMPs accounting for the complexity of the response of humans to venoms.     

Microcirculation and oxidative stress

The microcirculation is a complex and integrated system, transporting oxygen and nutrients to the cells. The key component of this system is the endothelium, contributing to the local balance between pro and anti-inflammatory mediators, hemostatic balance, as well as vascular permeability and cell proliferation. A constant shear stress maintains vascular endothelium homeostasis while perturbed shear stress leads to changes in secretion of vasodilator and vasoconstrictor agents. Increased oxidative stress is a major pathogenetic mechanism of endothelial dysfunction by decreasing NO bioavailability, promoting inflammation and participating in activation of intracellular signals cascade, so influencing ion channels activation, signal transduction pathways, cytoskeleton remodelling, intercellular communication and ultimately gene expression. Targeting the microvascular inflammation and oxidative stress is a fascinating approach for novel therapies in order to decrease morbidity and mortality of chronic and acute diseases.     

Oxidative stress in haemostasis

The normal hemostatic mechanisms consist of a balance between hemorrhage and thrombosis that is achieved through the interaction of the blood vessels, blood platelets, the coagulation and fibrinolytic factors. The vascular endothelium sustains the balance between prevention and stimulation of platelet activation, thrombogenesis and fibrinolysis and between vasoconstriction and vasodilatation. Endothelial dysfunction associated with different cardiovascular diseases is related to the local formation of reactive oxygen/nitrogen species, mainly peroxynitrite that is produced in a rapid reaction between nitric oxide and superoxide anion. Reactive oxygen/nitrogen species induce changes in the structure and function in hemostatic elements. Proteins and lipids are major initial targets in endothelial cells, blood platelets and plasma. Reaction of reactive oxygen species and nitrogen species, including peroxynitrite, with cellular proteins can lead to nitration of aromatic amino acid residues, oxidation of thiol groups and conversion of some amino acid residues into carbonyl derivative. Oxidative/nitrative modifications of platelet proteins may induce changes of their signaling and haemostatic function (activation). Peroxynitrite also causes oxidation and nitration of fibrinogen--a key protein in coagulation cascade and plasminogen (the main protein of fibrinolysisprocess) changing their hemostatic functions. Oxidative/nitrative modifications of different components of haemostasis system have been observed in several cardiovascular diseases.