Oral melatonin reduces blood coagulation activity: a placebo-controlled study in healthy young men

Melatonin has previously been suggested to affect hemostatic function but studies on the issue are scant. We hypothesized that, in humans, oral administration of melatonin is associated with decreased plasma levels of procoagulant hemostatic measures compared with placebo medication and that plasma melatonin concentration shows an inverse association with procoagulant measures. Forty-six healthy men (mean age 25 +/- 4 yr) were randomized, single-blinded, to either 3 mg of oral melatonin (n = 25) or placebo medication (n = 21). One hour thereafter, levels of melatonin, fibrinogen, and D-dimer as well as activities of coagulation factor VII (FVII:C) and VIII (FVIII:C) were measured in plasma. Multivariate analysis of covariance and regression analysis controlled for age, body mass index, mean arterial blood pressure, heart rate, and norepinephrine plasma level. Subjects on melatonin had significantly lower mean levels of FVIII:C (81%, 95% CI 71-92 versus 103%, 95% CI 90-119; P = 0.018) and of fibrinogen (1.92 g/L, 95% CI 1.76-2.08 versus 2.26 g/L, 95% CI 2.09-2.43; P = 0.007) than those on placebo explaining 14 and 17% of the respective variance. In all subjects, increased plasma melatonin concentration independently predicted lower levels of FVIII:C (P = 0.037) and fibrinogen (P = 0.022) explaining 9 and 11% of the respective variance. Melatonin medication and plasma concentration were not significantly associated with FVII:C and D-dimer levels. A single dose of oral melatonin was associated with lower plasma levels of procoagulant factors 60 min later. There might be a dose-response relationship between the plasma concentration of melatonin and coagulation activity.

Comparison of the in vitro effects of saline, hypertonic hydroxyethyl starch, hypertonic saline, and two forms of hydroxyethyl starch on whole blood coagulation and platelet function in dogs.

OBJECTIVE To compare the in vitro effects of hypertonic solutions and colloids to saline on coagulation in dogs. DESIGN In vitro experimental study. SETTING Veterinary teaching hospital. ANIMALS Twenty-one adult dogs. INTERVENTIONS Blood samples were diluted with saline, 7.2% hypertonic saline solution with 6% hydroxyethylstarch with an average molecular weight of 200 kDa and a molar substitution of 0.4 (HH), 7.2% hypertonic saline (HTS), hydroxyethyl starch (HES) 130/0.4 or hydroxyethyl starch 600/0.75 at ratios of 1:22 and 1:9, and with saline and HES at a ratio of 1:3. MEASUREMENTS AND MAIN RESULTS Whole blood coagulation was analyzed using rotational thromboelastometry (extrinsic thromboelastometry-cloting time (ExTEM-CT), maximal clot firmness (MCF) and clot formation time (CFT) and fibrinogen function TEM-CT (FibTEM-CT) and MCF) and platelet function was analyzed using a platelet function analyzer (closure time, CTPFA ). All parameters measured were impaired by saline dilution. The CTPFA was prolonged by 7.2% hypertonic saline solution with 6% hydroxyethylstarch with an average molecular weight of 200 kDa and a molar substitution of 0.4 (HH) and HTS but not by HES solutions. At clinical dilutions equivalent to those generally administered for shock (saline 1:3, HES 1:9, and hypertonic solutions 1:22), CTPFA was more prolonged by HH and HTS than other solutions but more by saline than HES. No difference was found between the HES solutions or the hypertonic solutions. ExTEM-CFT and MCF were impaired by HH and HTS but only mildly by HES solutions. At clinically relevant dilutions, no difference was found in ExTEM-CFT between HTS and saline or in ExTEM-MCF between HH and saline. No consistent difference was found between the 2 HES solutions but HH impaired ExTEM-CFT and MCF more than HTS. At high dilutions, FibTEM-CT and -MCF and ExTEM-CT were impaired by HES. CONCLUSIONS Hypertonic solutions affect platelet function and whole blood coagulation to a greater extent than saline and HES. At clinically relevant dilutions, only CTPFA was markedly more affected by hypertonic solutions than by saline. At high dilutions, HES significantly affects coagulation but to no greater extent than saline at clinically relevant dilutions.

In vitro effects of 3 % hypertonic saline and 20 % mannitol on canine whole blood coagulation and platelet function.

BACKGROUND: Hyperosmolar therapy, using either mannitol or hypertonic saline (HTS), is considered the treatment of choice for intracranial hypertension. However, hyperosmolar agents may impair coagulation and platelet function, limiting their use in patients at risk for hemorrhage. Despite this, studies evaluating the effects of mannitol compared to other hyperosmolar agents in dogs are largely lacking. The aim of this study was to compare the in vitro effects on global hemostasis and platelet function of 20 % mannitol and 3 % HTS on canine blood. METHODS: Citrated whole blood from 15 healthy dogs was diluted with 0.9 % saline, 20 % mannitol and 3 % HTS in ratios of 1:16 and 1:8. Rotational thromboelastometry (ROTEM) was used to assess clotting time (CT), clot formation time (CFT) and maximal clot firmness (MCF) following extrinsic activation (Ex-tem) and after platelet inhibition (Fib-tem). A platelet function analyzer (PFA-100) was used to assess closure time (CtPFA). RESULTS: No significant differences were observed between untreated whole blood and samples diluted with saline. Samples diluted with both mannitol and HTS were hypocoagulable compared to untreated whole blood samples. At a dilution of 1:16, no significant differences were found between any measured parameter in samples diluted with saline compared to mannitol or HTS. At a 1:8 dilution, CtPFA was prolonged in samples diluted with mannitol and HTS compared to saline, and CtPFA was prolonged more with mannitol than HTS. Ex-tem CT was increased at a 1:8 dilution with mannitol compared to HTS. Ex-tem CFT was prolonged at a 1:8 dilution with both agents compared to saline, and was prolonged more with mannitol than HTS. Ex-tem MCF was reduced at a 1:8 dilution with both agents compared to saline. DISCUSSION AND CONCLUSIONS: Data in this study indicate that both mannitol and HTS affect canine platelet function and whole blood coagulation in vitro in a dose-dependent fashion. The most pronounced effects were observed after high dilutions with mannitol, which impaired platelet aggregation, clot formation time, clot strength, and fibrin formation significantly more than HTS. Further in vivo studies are necessary before recommendations can be made

Structural basis for chemical inhibition of human blood coagulation factor Xa

Factor Xa, the converting enzyme of prothrombin to thrombin, has emerged as an alternative (to thrombin) target for drug discovery for thromboembolic diseases. An inhibitor has been synthesized and the crystal structure of the complex between Des[1–44] factor Xa and the inhibitor has been determined by crystallographic methods in two different crystal forms to 2.3- and 2.4-Å resolution. The racemic mixture of inhibitor FX-2212, (2RS)-(3′-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid, inhibits factor Xa activity by 50% at 272 nM in vitro. The S-isomer of FX-2212 (FX-2212a) was found to bind to the active site of factor Xa in both crystal forms. The biphenylamidine of FX-2212a occupies the S1-pocket, and the pyridine ring makes hydrophobic interactions with the factor Xa aryl-binding site. Several water molecules meditate inhibitor binding to residues in the active site. In contrast to the earlier crystal structures of factor Xa, such as those of apo-Des[1–45] factor Xa and Des[1–44] factor Xa in complex with a naphthyl inhibitor DX-9065a, two epidermal growth factor-like domains of factor Xa are well ordered in both our crystal forms as well as the region between the two domains, which recently was found to be the binding site of the effector cell protease receptor-1. This structure provides a basis for designing next generation inhibitors of factor Xa.

Fatal embryonic bleeding events in mice lacking tissue factor, the cell-associated initiator of blood coagulation.

Tissue factor (TF) is the cellular receptor for coagulation factor VI/VIIa and is the membrane-bound glycoprotein that is generally viewed as the primary physiological initiator of blood coagulation. To define in greater detail the physiological role of TF in development and hemostasis, the TF gene was disrupted in mice. Mice heterozygous for the inactivated TF allele expressed approximately half the TF activity of wild-type mice but were phenotypically normal. However, homozygous TF-/- pups were never born in crosses between heterozygous mice. Analysis of mid-gestation embryos showed that TF-/- embryos die in utero between days 8.5 and 10.5. TF-/- embryos were morphologically distinct from their TF+/+ and TF+/- littermates after day 9.5 in that they were pale, edematous, and growth retarded. Histological studies showed that early organogenesis was normal. The initial failure in TF-/- embryos appeared to be hemorrhaging, leading to the leakage of embryonic red cells from both extraembryonic and embryonic vessels. These studies indicate that TF plays an indispensable role in establishing and/or maintaining vascular integrity in the developing embryo at a time when embryonic and extraembryonic vasculatures are fusing and blood circulation begins.

Mathematical analysis of activation thresholds in enzyme-catalyzed positive feedbacks: application to the feedbacks of blood coagulation.

A hierarchy of enzyme-catalyzed positive feedback loops is examined by mathematical and numerical analysis. Four systems are described, from the simplest, in which an enzyme catalyzes its own formation from an inactive precursor, to the most complex, in which two sequential feedback loops act in a cascade. In the latter we also examine the function of a long-range feedback, in which the final enzyme produced in the second loop activates the initial step in the first loop. When the enzymes generated are subject to inhibition or inactivation, all four systems exhibit threshold properties akin to excitable systems like neuron firing. For those that are amenable to mathematical analysis, expressions are derived that relate the excitation threshold to the kinetics of enzyme generation and inhibition and the initial conditions. For the most complex system, it was expedient to employ numerical simulation to demonstrate threshold behavior, and in this case long-range feedback was seen to have two distinct effects. At sufficiently high catalytic rates, this feedback is capable of exciting an otherwise subthreshold system. At lower catalytic rates, where the long-range feedback does not significantly affect the threshold, it nonetheless has a major effect in potentiating the response above the threshold. In particular, oscillatory behavior observed in simulations of sequential feedback loops is abolished when a long-range feedback is present.

Tissue factor promotes melanoma metastasis by a pathway independent of blood coagulation.

Several studies have established a link between blood coagulation and cancer, and more specifically between tissue factor (TF), a transmembrane protein involved in initiating blood coagulation, and tumor metastasis. In the study reported here, a murine model of human melanoma metastasis was used for two experiments. (i) The first experiment was designed to test the effect of varying the level of TF expression in human melanoma cells on their metastatic potential. Two matched sets of cloned human melanoma lines, one expressing a high level and the other a low level of the normal human TF molecule, were generated by retroviral-mediated transfections of a nonmetastatic parental line. The metastatic potential of the two sets of transfected lines was compared by injecting the tumor cells into the tail vein of severe combined immunodeficiency (SCID) mice and later examining the lungs and other tissues for tumor development. Metastatic tumors were detected in 86% of the mice injected with the high-TF lines and in 5% of the mice injected with the low-TF lines, indicating that a high TF level promotes metastasis of human melanoma in the SCID mouse model. This TF effect on metastasis occurs with i.v.-injected melanoma cells but does not occur with primary tumors formed from s.c.-injected melanoma cells, suggesting that TF acts at a late stage of metastasis, after tumor cells have escaped from the primary site and entered the blood. (ii) The second experiment was designed to analyze the mechanism by which TF promotes melanoma metastasis. The procedure involved testing the effect on metastasis of mutations in either the extracellular or cytoplasmic domains of the transmembrane TF molecule. The extracellular mutations introduced two amino acid substitutions that inhibited initiation by TF of the blood-coagulation cascade; the cytoplasmic mutation deleted most of the cytoplasmic domain without impairing the coagulation function of TF. Several human melanoma lines expressing high levels of either of the two mutant TF molecules were generated by retroviral-mediated transfection of the corresponding TF cDNA into the nonmetastatic parental melanoma line, and the metastatic potential of each transfected line was tested in the SCID mouse model. Metastases occurred in most mice injected with the melanoma lines expressing the extracellular TF mutant but were not detected in most mice injected with the melanoma lines expressing the cytoplasmic TF mutant. Results with the extracellular TF mutant indicate that the metastatic effect of TF in the SCID mouse model does not involve products of the coagulation cascade. Results with the cytoplasmic TF mutant indicate that the cytoplasmic domain of TF is important for the metastatic effect, suggesting that the TF could transduce a melanoma cell signal that promotes metastasis.

Human blood coagulation and its disorders,

Bibliography: p. 369-398.

Use of first nucleotide change technology to determine the frequency of factor V Leiden in a population of Australian blood donors

Activated protein C resistance (APCR), the most common risk factor for venous thrombosis, is the result of a G to A base substitution at nucleotide 1691 (R506Q) in the factor V gene. Current techniques to detect the factor V Leiden mutation, such as determination of restriction length polymorphisms, do not have the capacity to screen large numbers of samples in a rapid, cost- effective test. The aim of this study was to apply the first nucleotide change (FNC) technology, to the detection of the factor V Leiden mutation. After preliminary amplification of genomic DNA by polymerase chain reaction (PCR), an allele-specific primer was hybridised to the PCR product and extended using fluorescent terminating dideoxynucleotides which were detected by colorimetric assay. Using this ELISA-based assay, the prevalence of the factor V Leiden mutation was determined in an Australian blood donor population (n = 500). A total of 18 heterozygotes were identified (3.6%) and all of these were confirmed with conventional MnlI restriction digest. No homozygotes for the variant allele were detected. We conclude from this study that the frequency of 3.6% is compatible with others published for Caucasian populations. In addition, the FNC technology shows promise as the basis for a rapid, automated DNA based test for factor V Leiden.

Controlling whole blood activation and resultant clot properties on various material surfaces : a possible therapeutic approach for enhancing bone healing

Injured bone initiates the healing process by forming a blood clot at the damaged site. However, in severe damage, synthetic bone implants are used to provide structural integrity and restore the healing process. The implant unavoidably comes into direct contact with whole blood, leading to a blood clot formation on its surface. Despite this, most research in bone tissue engineering virtually ignores the important role of a blood clot in supporting healing. Surface chemistry of a biomaterial is a crucial property in mediating blood-biomaterials interactions, and hence the formation of the resultant blood clot. Surfaces presenting mixtures of functional groups carboxyl (–COOH) and methyl (–CH3) have been shown to enhance platelet response and coagulation activation, leading to the formation of fibrin fibres. In addition, it has been shown that varying the compositions of these functional groups and the length of alkyl groups further modulate the immune complement response. In this study, we hypothesised that a biomaterial surface with mixture of –COOH/–CH3(methyl), –CH2CH3 (ethyl) or –(CH2)3CH3 (butyl) groups at different ratios would modulate blood coagulation and complement activation, and eventually tailor the structural and functional properties of the blood clot formed on the surface, which subsequently impacts new bone formation. Firstly, we synthesised a series of materials composed of acrylic acid (AA), and methyl (MMA), ethyl (EMA) or butyl methacrylates (BMA) at different ratios and coated on the inner surfaces of incubation vials. Our surface analysis showed that the amount of –COOH groups on the surface coatings was lower than the ratios of AA prepared in the materials even though the surface content of –COOH groups increased with increasing in AA ratios. It was indicated that the surface hydrophobicity increased with increasing alkyl chain length: –CH 3 > –CH2CH3 > –(CH2)3CH3, and decreased with increasing –COOH groups. No significant differences in surface hydrophobicity was found on surfaces with –CH3 and –CH2CH3 groups in the presence of –COOH groups. The material coating was as smooth as uncoated glass and without any major flaws. The average roughness of material-coated surface (3.99 ± 0.54 nm) was slightly higher than that of uncoated glass surface (2.22 ± 0.29 nm). However, no significant differences in surface average roughness was found among surfaces with the same functionalities at different –COOH ratios nor among surfaces with different alkyl groups but the same –COOH ratios. These suggested that the surface functional groups and their compositions had a combined effect on modulating surface hydrophobicity but not surface roughness. The second part of our study was to investigate the effect of surface functional groups and their compositions on blood cascade activation and structural properties of the formed clots. It was found that surfaces with –COOH/–(CH2)3CH3 induced a faster coagulation activation than those with –COOH/–CH3 and –CH2CH3, regardless of the –COOH ratios. An increase in –COOH ratios on –COOH/–CH3 and –CH2CH3 surfaces decreased the rate of activation. Moreover, all material-coated surfaces markedly reduced the complement activation compared to uncoated glass surfaces, and the pattern of complement activation was entirely similar to that of surface-induced coagulation, suggesting there is an interaction between two cascades. The clots formed on material-coated surfaces had thicker fibrin with a tighter network at the exterior when compared to uncoated glass surfaces. Compared to the clot exteriors, thicker fibrins with a loose network were found in clot interiors. Coated surfaces resulted in more rigid clots with a significantly slower fibrinolysis after 1 h of lysis when compared to uncoated glass surfaces. Significant differences in fibrinolysis after 1 h of lysis among clots on material-coated surfaces correlated well with the differences in fibrin thickness and density at clot exterior. In addition, more growth factors were released during clot formation than during clot lysis. From an intact clot, there was a correlation between the amount of PDGF-AB release and fibrin density. Highest amount of PDGF-AB was released from clots formed on surfaces with 40% –COOH/60% –CH 3 (i.e. 65MMA). During clot lysis, the release of PDGF-AB also correlated with the fibrinolytic rate while the release of TGF-â1 was influenced by the fibrin thickness. This suggested that different clot structures led to different release profiles of growth factors in clot intact and degrading stages. We further validated whether the clots formed on material-coatings provide the microenvironment for improved bone healing by using a rabbit femoral defect model. In this pilot study, the implantation of clots formed on 65MMA coatings significantly increased new bone formation with enhanced chondrogenesis, osteoblasts activity and vascularisation, but decreased inflammatory macrophage number at the defects after 4 weeks when compared to commercial bone grafts ChronOSTM â-TCP granules. Empty defects were observed when blood clot formation was inhibited. In summary, our study demonstrated that surface functional groups and their relative ratios on material coatings synergistically modulate activation of blood cascades, resultant fibrin architecture, rigidity, susceptibility to fibrinolysis as well as growth factor release of the formed clots, which ultimately alter the healing microenvironment of injured bones.

Controlling whole blood activation and resultant clot properties by carboxyl and alkyl functional groups on material surfaces : a possible therapeutic approach for enhancing bone healing

Most research virtually ignores the important role of a blood clot in supporting bone healing. In this study, we investigated the effects of surface functional groups carboxyl and alkyl on whole blood coagulation, complement activation and blood clot formation. We synthesised and tested a series of materials with different ratios of carboxyl (–COOH) and alkyl (–CH3, –CH2CH3 and –(CH2)3CH3) groups. We found that surfaces with –COOH/–(CH2)3CH3 induced a faster coagulation activation than those with –COOH/– CH3 and –CH2CH3, regardless of the –COOH ratios. An increase in –COOH ratios on –COOH/–CH3 and –CH2CH3 surfaces decreased the rate of coagulation activation. The pattern of complement activation was entirely similar to that of surface-induced coagulation. All material coated surfaces resulted in clots with thicker fibrin in a denser network at the clot/material interface and a significantly slower initial fibrinolysis when compared to uncoated glass surfaces. The amounts of platelet-derived growth factor-AB (PDGF-AB) and transforming growth factor-b (TGF-b1) released from an intact clot were higher than a lysed clot. The release of PDGF-AB was found to be correlated with the fibrin density. This study demonstrated that surface chemistry can significantly influence the activation of blood coagulation and complement system, resultant clot structure, susceptibility to fibrinolysis as well as release of growth factors, which are important factors determining the bone healing process.

Formation of blood clot on biomaterial implants influences bone healing

The first step in bone healing is forming a blood clot at injured bones. During bone implantation, biomaterials unavoidably come into direct contact with blood, leading to a blood clot formation on its surface prior to bone regeneration. Despite both situations being similar in forming a blood clot at the defect site, most research in bone tissue engineering virtually ignores the important role of a blood clot in supporting healing. Dental implantology has long demonstrated that the fibrin structure and cellular content of a peri-implant clot can greatly affect osteoconduction and de novo bone formation on implant surfaces. This paper reviews the formation of a blood clot during bone healing in related to the use of platelet-rich plasma (PRP) gels. It is implicated that PRP gels are dramatically altered from a normal clot in healing, resulting conflicting effect on bone regeneration. These results indicate that the effect of clots on bone regeneration depends on how the clots are formed. Factors that influence blood clot structure and properties in related to bone healing are also highlighted. Such knowledge is essential for developing strategies to optimally control blood clot formation, which ultimately alter the healing microenvironment of bone. Of particular interest are modification of surface chemistry of biomaterials, which displays functional groups at varied composition for the purpose of tailoring blood coagulation activation, resultant clot fibrin architecture, rigidity, susceptibility to lysis, and growth factor release. This opens new scope of in situ blood clot modification as a promising approach in accelerating and controlling bone regeneration.