Reduced Bleeding Time Research Articles

Antifibrinolytic therapy to reduce haemoptysis from any cause.

Haemoptysis is a common pathology around the world, occurring with more frequency in low-income countries. It has different etiologies, many of which have infectious characteristics. Antifibrinolytic agents are commonly used to manage bleeding from different sources, but their usefulness in pulmonology is unclear. To evaluate the effectiveness and safety of antifibrinolytic agents in reducing the volume and duration of haemoptysis in adult and paediatric patients. We searched the Cochrane Central Register of Controlled Trials (CENTRAL) and the Database of Abstracts of Reviews of Effects (DARE) in The Cochrane Library, EMBASE and LILACS for publications that describe randomized controlled trials (RCTs) of antifibrinolytic therapy in patients presenting with haemoptysis. We also performed an independent search in MEDLINE for relevant trials not yet included in CENTRAL or DARE. Searches are up to date to the 19th September 2016. We conducted electronic and manual searches of relevant national and international journals. We reviewed the reference lists of included studies to locate relevant randomized controlled trials (RCTs). An additional search was carried out to find unpublished RCTs. We included RCTs designed to evaluate the effectiveness and safety of antifibrinolytic agents in reducing haemoptysis in adult and paediatric patients of both genders presenting with haemoptysis of any etiology and severity. The intervention of interest was the administration of antifibrinolytic agents compared with placebo or no treatment. All reviewers independently assessed methodological quality and extracted data tables pre-designed for this review. The electronic literature search identified 1 original study that met the eligibility criteria. One unpublished study was also identified through manual searches. Therefore two randomized controlled trials met the inclusion criteria: Tscheikuna 2002 (via electronic searches) and Ruiz 1994 (via manual searches). Tscheikuna 2002, a double-blind RCT performed in Thailand, evaluated the effectiveness of tranexamic acid (TXA, an antifibrinolytic agent) administered orally in 46 hospital in- and outpatients with haemoptysis of various etiologies. Ruiz 1994, a double-blind RCT performed in Peru, evaluated the effectiveness of intravenous TXA in 24 hospitalised patients presenting with haemoptysis secondary to tuberculosis.Pooled together, results demonstrated a significant reduction in bleeding time between patients receiving TXA and patients receiving placebo with a weighted mean difference (WMD) of -19.47 (95% CI -26.90 to -12.03 hours), but with high heterogeneity (I² = 52%). TXA did not affect remission of haemoptysis evaluated at seven days after the start of treatment. Adverse effects caused by the drug's mechanism of action were not reported. There was no significant difference in the incidence of mild side effects between active and placebo groups (OR 3.13, 95% CI 0.80 to 12.24). There is insufficient evidence to judge whether antifibrinolytics should be used to treat haemoptysis from any cause, though limited evidence suggests they may reduce the duration of bleeding.

Facile Assembly of Cost-Effective and Locally Applicable or Injectable Nanohemostats for Hemorrhage Control.

Currently, there is still unmet demand for effective and safe hemostats to control abnormal bleeding in different conditions. With the aim to develop affordable, safe, effective, easily stored, and low-cost hemostats, we developed a series of positively charged nanoparticles by a facile one-pot assembly approach. In this strategy, nanoparticles were formed by cholic-acid-mediated self-assembly of polyethylenimine (PEI). Regardless of different structures of cholic acids and PEIs, well-defined nanoparticles could be successfully formed. The assembly process was dominated by multiple interactions between cholic acid and PEI, including electrostatic, hydrogen bonding, and hydrophobic forces. In vitro studies showed that assembled nanoparticles effectively induced aggregation and activation of platelets. Local application of aqueous solution containing nanoparticles assembled by different cholic acids and PEIs significantly reduced bleeding times in different rodent models including tail transection in mice as well as liver bleeding and femoral artery bleeding in rats or rabbits. Moreover, intravenous (i.v.) injection of this type of positively charged nanoparticles notably prevented bleeding in the femoral artery in rats by targeting the injured site via opsonization of nanoparticles with fibrinogen. By contrast, a control negatively charged nanoparticle showed no hemostatic activity after i.v. delivery. Also, preliminary evaluations in rats revealed a good safety profile after i.v. administration of assembled nanoparticles at a dose 4-fold higher than that used for hemostasis. These results demonstrated that cholic acid/PEI-assembled positive nanoparticles may function as cost-effective and locally applicable or injectable nanohemostats for hemorrhage control in the civilian setting and on the battlefield.

Proteome analysis of mast cell releasates reveals a role for chymase in the regulation of coagulation factor XIIIA levels via proteolytic degradation

Mast cells are significantly involved in IgE-mediated allergic reactions; however, their roles in health and disease are incompletely understood. We aimed to define the proteome contained in mast cell releasates on activation to better understand the factors secreted by mast cells that are relevant to the contribution of mast cells in diseases. Bone marrow-derived cultured mast cells (BMCMCs) and peritoneal cell-derived mast cells were used as "surrogates" for mucosal and connective tissue mast cells, respectively, and their releasate proteomes were analyzed by mass spectrometry. Our studies showed that BMCMCs and peritoneal cell-derived mast cells produced substantially different releasates following IgE-mediated activation. Moreover, we observed that the transglutaminase coagulation factor XIIIA (FXIIIA) was one of the most abundant proteins contained in the BMCMC releasates. Mast cell-deficient mice exhibited increased FXIIIA plasma and activity levels as well as reduced bleeding times, indicating that mast cells are more efficient in their ability to downregulate FXIIIA than in contributing to its amounts and functions in homeostatic conditions. We found that human chymase and mouse mast cell protease-4 (the mouse homologue of human chymase) had the ability to reduce FXIIIA levels and function via proteolytic degradation. Moreover, we found that chymase deficiency led to increased FXIIIA amounts and activity, as well as reduced bleeding times in homeostatic conditions and during sepsis. Our study indicates that the mast cell protease content can shape its releasate proteome. Moreover, we found that chymase plays an important role in the regulation of FXIIIA via proteolytic degradation.

Hemostatic activities of nano/microporous bilayer dressings in a femoral artery bleeding rat model

ABSTRACTRecently, an effective hemostatic dressing requirement has become a major problem in both the military and civilian world. Available hemostatic agents are too expensive, ineffective, unsafe, or complicated to use. Here, we evaluated the hemostatic efficacy of a nano/micro bilayer hemostatic dressing including a porous sublayer from chitosan (CTS) and bacterial cellulose (BC) and a nanofibrillar upper layer from silk fibroin (SF). In addition, several active agents rolled in coagulation cascade [vitamin K (Vit K), protamine sulfate (PS), kaolin (Kao), etc.] were doped to the sublayer of bilayer hemostatic agent and their activities were compared via in vivo and in vitro tests. Lactate dehydrogenase (LDH) activity test results demonstrated that BC/CTS, SF‐coated BC/CTS, and Vit K K/BC/CTS, SF/phosphatidylcholine (PC)‐coated BC/CTS showed higher LDH activity compared to standard gauze (p < 0.005). In a femoral artery bleeding rat model, SF‐coated PS/BC/CTS significantly reduced bleeding time (80 ± 0.3 s) compared to standard gauze and kaolin‐doped group (p < 0.005). Blood loss and mortality rate with 2.3 g and 37.5% SF‐coated BC/CTS and SF‐coated PS/BC/CTS showed increased efficacy in achieving hemostasis compared to standard gauze. All the prepared hemostatic dressings except for kaolin‐doped BC/CTS and standard gauze induced no inflammatory reaction in the tissue. All these data suggest that active agent‐doped BC/CTS/SF‐based bilayer hemostatic dressings have a great influence on the resulting hemostatic action. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43657.

Effects evaluation of rapid response first-aid case on bedside emergency endoscopic hemostasis therapy

Objective To explore the effects of rapid response first-aid case on bedside emergency gastroscopy hemostatic treatment. Methods A total of 52 patients with esophageal-fundal varices in liver cirrhosis from January 2014 to June 2015 were divided into control group (n=25) and experimental group (n=27). The patients of control group received the routine nursing; then we summarized the experience and mistakes of operation cooperation, optimized process; next we designed rapid response first-aid case and used in the experimental group. The blood transfusion time, goods preparation time, blood loss, blood transfusion volume were compared in two groups. Results The data of the experimental group in average bleeding to transfusion time (90.22±36.47)min, materials preparation time (14.66±3.48) min, the average amount of bleeding (369.62±158.90) ml, average blood transfusion amount (322.40±117.82) ml were lower than those of the control group [average bleeding to blood transfusion time (123.60±51.87) min, materials preparation time (22.44±2.59) min, average bleeding volume (660.20±181.82) ml, average blood transfusion amount (458.00±140.63)ml](P<0.05). Conclusions The implementation of rapid response first-aid case can effectively shorten the rescue time, reduce bleeding and blood transfusion time, the blood loss, the wastage of human resources and medical resources, improve the efficiency of nursing work in the department and the success rate of rescue. Key words: First aid; Endoscopy; Hemostasis; Rapid response first-aid case

Transfusion with synthetic platelet substitutes, H12-(ADP)liposomes, effectively rescues thrombocytopenic rabbits from massive hemorrhage

Event Abstract Back to Event Transfusion with synthetic platelet substitutes, H12-(ADP)liposomes, effectively rescues thrombocytopenic rabbits from massive hemorrhage Masato Takikawa1, Kosuke Hagisawa2, Manabu Kinoshita3, Haruka Asahina4, Daizo Saito5 and Shinji Takeoka1 1 Waseda University, Graduate School of Advanced Science and Engineering, Japan 2 National Defense Medical College, Department of Physiology, Japan 3 National Defense Medical College, Department of Immunology and Microbioogy, Japan 4 National Defense Medical College, Department of Traumatology and Critical Care Medicine, Japan 5 National Defense Medical College Research Institute, Division of Traumatology, Japan Introduction: Hemorrhagic shock and the following coagulopathy are critical conditions in traumatic patients[1]. Resuscitative transfusion composed of plasma, red blood cells (RBCs) and platelets (PLTs) ratios of 1:1:1 was reported to be effective in rescuing such fatally injured patients. Although PLT transfusion is desirable to treat the coagulopathy, it is restricted in particular prehospital resuscitation due to its short shelf life. In order to address this crucial issue, we have developed H12-(ADP)liposomes as a liposome-type PLTs substitute[2]. H12-(ADP)liposomes are conjugated with dodecapeptides (H12, HHLGGAKQAGDV), which is a carboxy terminal sequence of fibrinogen gamma-chain, on liposomes’ surface, and encapsulated with adenosine 5’-phosphate (ADP), which enhances PLT aggregation. We investigated a hemostatic effect of H12-(ADP)liposomes on the massive hemorrhage in thrombocytopenic rabbits. Materials and Methods: Sixteen male NZW rabbits weighed 2.4 kg were used. Thrombocytopenia (PLT count; approximately 46×103/μL) was induced in the rabbits by repeated blood withdrawal and isovolemic transfusion of autologous washed RBCs. Thereafter, a liver injury was made followed by balloon compression for 5 min. During balloon compression, H12-(ADP)liposomes/PPP (Platelet-Poor Plasma) (n=6), PRP (Platelet-Rich Plasma) (n=6), PPP (n=4) was transfused to the rabbits. After the removal of compression, bleeding volume from the injured site was measured for the initial 10 min. Bleeding time was also monitored in the subsequent 120 min. After achieving hemostasis, transfusion of RBCs from other rabbits ([Hb]≒17.1 g/dL) was performed to recover from the severe hypotension and hypohemoglobinemia. Thereafter, their survivals were observed for 48 hours. Results: Rabbits in the H12-(ADP)liposomes/PPP transfusion group stopped bleeding at 47 min after liver injury (PRP group stopped at 37 min), and their bleeding volume for 10 min after liver injury was 18.9 g (PRP group; 19.4 g). None of the rabbits in PPP group was able to achieve hemostasis and thereby survived beyond 3 hours. RBCs transfusion after achieving hemostasis raised MAP from 53 to 59 mmHg in H12-(ADP)liposomes/PPP and PRP groups. Survival rates of H12-(ADP)liposomes/PPP and PRP groups were 83%. Discussions: Transfusion with H12-(ADP)liposomes/PPP as well as PRP effectively reduced bleeding time and volume in rabbits comparing to the transfusion with PPP. There were no significant differences in bleeding time/volume between these two groups. Conclusion: Thus, H12-(ADP)liposomes may have a potent hemostatic effect and thereby improve the survival rate that is equivalent to PRP.

Modulation of cGMP/cAMP crosstalk on the level of PDE3 in animals lacking NO-sensitive guanylyl cyclase

NO-sensitive guanylyl cyclase (NO-GC) is accepted to be the major receptor for the signaling molecule NO. Deletion of NO-GC in mice leads to disturbed NO/cGMP signaling. As a result, these mice show abolished NO-dependent relaxation of smooth muscle-containing tissues in the cardiovascular and gastrointestinal systems. Mice with general deletion (GCKO) suffer from increased blood pressure, reduced bleeding time, erectile dysfunction and die prematurely due to gastrointestinal dysmotility. Mice lacking NO-GC specifically in smooth muscle cells (SMC) show an elevated blood pressure similar to that seen in GCKO animals. Crosstalk between cGMP and cAMP signaling has been reported to occur in many different cells. Phosphodiesterase 3 (PDE3) is thought to be one of the prominent mediators of this crosstalk. Through its inhibitory action on PDE3, cGMP may regulate cAMP levels and cAMP-mediated signaling. Here, we reasoned that, in animals deficient in NO-GC, lack of NO-induced cGMP synthesis might affect cGMP/cAMP crosstalk on the level of PDE3. Smooth muscle relaxation induced by forskolin to stimulate adenylyl cyclase was similar in WT, GCKO and SMC-GCKO aortae. RIA-based detection of basal cAMP levels in aortic tissue from these mice did not reveal differences. Similarly, FRET measurements showed that PGE1-induced cAMP synthesis in isolated smooth muscle cells was unaffected by NO-GC deletion. However, aortae from GCKO and SMC-GCKO animals were more sensitive to milrinone, a specific inhibitor of PDE3, as the concentration curve for relaxation was shifted to the left. Analysis of PDE3 expression in aorta showed an approximately 50% reduction in PDE3 protein and specific enzyme activity. Similar reduction was detected in platelets but not in heart tissue of GCKO animals. Induction of SMC-specific NO-GC deletion by tamoxifen led to a parallel decline of NO-GC and PDE3 expression. Both proteins were strongly reduced already 5 days after the last tamoxifen injection. In contrast, systolic blood pressure in these animals started to rise only after 10 days and was maximal after 50 days. These data indicate that cGMP, not the increase in blood pressure, is responsible for the expression regulation of PDE3. We conclude that cGMP modulates PDE3 expression in SMC in order to keep up functional cAMP signaling.

Tranexamic Acid for Epistaxis--A Promising Treatment That Deserves Further Study.

Does the application of topical tranexamic acid reduce bleeding as compared to anterior packing? Zahed R, Moharamzadeh P, Alizadeharasi S, et al. A new and rapid method for epistaxis treatment using injectable form of tranexamic acid topically: a randomized controlled trial. Am J Emerg Med 2013;31(9):1389-92. To determine if topically applied tranexamic acid reduces bleeding time in epistaxis.

Combined genetic effects of EGLN1 and VWF modulate thrombotic outcome in hypoxia revealed by Ayurgenomics approach.

BackgroundExtreme constitution “Prakriti” types of Ayurveda exhibit systemic physiological attributes. Our earlier genetic study has revealed differences in EGLN1, key modulator of hypoxia axis between Prakriti types. This was associated with differences in high altitude adaptation and susceptibility to high altitude pulmonary edema (HAPE). In this study we investigate other molecular differences that contribute to systemic attributes of Prakriti that would be relevant in predictive marker discovery.MethodsGenotyping of 96 individuals of the earlier cohort was carried out in a panel of 2,800 common genic SNPs represented in Indian Genomic Variation Consortium (IGVC) panel from 24 diverse populations. Frequency distribution patterns of Prakriti differentiating variations (FDR correction P < 0.05) was studied in IGVC and 55 global populations (HGDP–CEPH) panels. Genotypic interactions between VWF, identified from the present analysis, and EGLN1 was analyzed using multinomial logistic regression in Prakriti and Indian populations from contrasting altitudes. Spearman’s Rank correlation was used to study this genotypic interaction with respect to altitude in HGDP–CEPH panel. Validation of functional link between EGLN1 and VWF was carried out in a mouse model using chemical inhibition and siRNA studies.ResultSignificant differences in allele frequencies were observed in seven genes (SPTA1, VWF, OLR1, UCP2, OR6K3, LEPR, and OR10Z1) after FDR correction (P < 0.05). A non synonymous variation (C/T, rs1063856) associated with thrombosis/bleeding susceptibility respectively, differed significantly between Kapha (C-allele) and Pitta (T-allele) constitution types. A combination of derived EGLN1 allele (HAPE associated) and ancestral VWF allele (thrombosis associated) was significantly high in Kapha group compared to Pitta (p < 10–5). The combination of risk-associated Kapha alleles was nearly absent in natives of high altitude. Inhibition of EGLN1 using (DHB) and an EGLN1 specific siRNA in a mouse model lead to a marked increase in vWF levels as well as pro-thrombotic phenotype viz. reduced bleeding time and enhanced platelet count and activation.ConclusionWe demonstrate for the first time a genetic link between EGLN1 and VWF in a constitution specific manner which could modulate thrombosis/bleeding susceptibility and outcomes of hypoxia. Integration of Prakriti in population stratification may help assemble common variations in key physiological axes that confers differences in disease occurrence and patho-phenotypic outcomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0542-9) contains supplementary material, which is available to authorized users.

Pentadecapeptide BPC 157 Reduces Bleeding and Thrombocytopenia after Amputation in Rats Treated with Heparin, Warfarin, L-NAME and L-Arginine.

BackgroundBPC 157 is a stable gastric pentadecapeptide recently implicated with a role in hemostasis. While NO is largely implicated in hemostatic mechanisms, in tail-amputation-models under heparin- and warfarin-administration, both the NO-synthase (NOS)-blocker, L-NAME (prothrombotic) and the NOS-substrate L-arginine (antithrombotic), were little investigated. Objective. To investigate the effect of L-NAME and L-arginine on hemostatic parameters, and to reveal the effects of BPC 157 on the L-NAME- and L-arginine-induced hemostatic actions under different pathological condition: tail amputation without or with anticoagulants, heparin or warfarin.MethodsTail amputation, and/or i.v.-heparin (10 mg/kg), i.g.-warfarin (1.5 mg/kg/day for 3 days) were used in rats. Treatment includes BPC 157, L-NAME, L-arginine, per se and their combination.ResultsAfter (tail) amputation, with or without i.v.-heparin or i.g.-warfarin, BPC 157 (10 μg/kg, 10 ng/kg, i.p., i.v. (heparin), 10 μg/kg i.g. (warfarin)) always reduced bleeding time and/or haemorrhage and counteracted thrombocytopenia. As for L-NAME and/or L-arginine, we noted: L-arginine (100 mg/kg i.p.)–rats: more bleeding, less/no thrombocytopenia; L-NAME (5 mg/kg i.p.)-rats: less bleeding (amputation only), but present thrombocytopenia; L-NAME+L-arginine-rats also exhibited thrombocytopenia: L-NAME counteracted L-arginine-increased bleeding, L-arginine did not counteract L-NAME-thrombocytopenia. All animals receiving BPC 157 in addition (BPC 157μg+L-NAME; BPC 157μg+L-arginine, BPC 157μg+L-NAME+L-arginine), exhibited decreased haemorrhage and markedly counteracted thrombocytopenia.ConclusionsL-NAME (thrombocytopenia), L-arginine (increased haemorrhage) counteraction and BPC 157 (decreased haemorrhage, counteracted thrombocytopenia) with rescue against two different anticoagulants, implicate a BPC 157 modulatory and balancing role with rescued NO-hemostatic mechanisms.

Highlights from the latest articles in nano-oncology.

Highlights from the latest articles in nano-oncology.

The Clot Thickens…

Ultra-deformable synthetic platelets stop bleeding in minutes.

Platelet-like nanoparticles: mimicking shape, flexibility, and surface biology of platelets to target vascular injuries.

Targeted delivery of therapeutic and imaging agents in the vascular compartment represents a significant hurdle in using nanomedicine for treating hemorrhage, thrombosis, and atherosclerosis. While several types of nanoparticles have been developed to meet this goal, their utility is limited by poor circulation, limited margination, and minimal targeting. Platelets have an innate ability to marginate to the vascular wall and specifically interact with vascular injury sites. These platelet functions are mediated by their shape, flexibility, and complex surface interactions. Inspired by this, we report the design and evaluation of nanoparticles that exhibit platelet-like functions including vascular injury site-directed margination, site-specific adhesion, and amplification of injury site-specific aggregation. Our nanoparticles mimic four key attributes of platelets, (i) discoidal morphology, (ii) mechanical flexibility, (iii) biophysically and biochemically mediated aggregation, and (iv) heteromultivalent presentation of ligands that mediate adhesion to both von Willebrand Factor and collagen, as well as specific clustering to activated platelets. Platelet-like nanoparticles (PLNs) exhibit enhanced surface-binding compared to spherical and rigid discoidal counterparts and site-selective adhesive and platelet-aggregatory properties under physiological flow conditions in vitro. In vivo studies in a mouse model demonstrated that PLNs accumulate at the wound site and induce ∼65% reduction in bleeding time, effectively mimicking and improving the hemostatic functions of natural platelets. We show that both the biochemical and biophysical design parameters of PLNs are essential in mimicking platelets and their hemostatic functions. PLNs offer a nanoscale technology that integrates platelet-mimetic biophysical and biochemical properties for potential applications in injectable synthetic hemostats and vascularly targeted payload delivery.

Abstract 262: Mitochondrial N-formyl Peptides Lead to Sepsis-like Symptoms via Basophil Activation

Traumatic injury may lead to bacteria-induced systemic inflammation (sepsis). However, 15% of trauma patients present sepsis-like symptoms in the absence of infection. In these patients, it is possible that fragments from mitochondria (damage-associated molecular patterns) from traumatized tissue act to initiate an inflammatory response. Previously, we have demonstrated that infusion of mitochondrial N-formyl peptides (F-MIT) leads to exacerbated vasodilatation and hypotension via formly-preptide receptor (FPR) activation and inducible nitric oxide release. However, the cell type mediating this effect was not determined. We hypothesized that F-MIT induces sepsis-like symptoms, include hypotension, via basophil activation. Male Wistar rats (12-weeks old, n=5-8) were cannulated (femoral artery and vein) for blood pressure recordings and drug infusion. To analyze organ damage, the lungs were collected and stained with H&amp;E. F-MIT infusion (0.02 mg/rat/ i.v. ) induced severe lung injury (edema, neuthrophil infiltration and alveolar thickening) (4-fold vs. control) and vascular leakage in aorta (+34%) and mesenteric resistance arteries (+66%). Moreover, F-MIT infusion increased body temperature (Control: 36±0.09 vs. F-MIT: 37±0.28 °C, *p&lt;0.05) and reduced bleeding time (Control: 89±8 vs. F-MIT: 33±11 seconds, *p&lt;0.05). Since F-MIT induced vascular leakage, rats were treated with histamine 2 receptor antagonist, cimetidine (50 mg/kg/ i.v. ) prior F-MIT infusion. As expected, F-MIT (0.02 mg/rat) induced hypotension [Control: 82±3 vs. F-MIT: 60±4 mmHg, *p&lt;0.05]. However, cimetidine treatment abolished this response (Control: 75±1 vs. F-MIT infusion: 70±4 mmHg). Interesting, F-MIT had no effect on blood pressure in animals treated with anti-asialo GM1 antiserum (0.2 ml/ i.p .) for depletion of basophils. Neither neutrophil nor mast cell depletion blocked F-MIT-induced hypotension (p&gt;0.05). These data suggest that F-MIT, via basophil activation, leads to histamine release, vascular leakage and hypotension, associated to organ damage, fever, blood clotting. Thus, F-MIT may be the link between SIRS and cardiovascular collapse in sterile trauma patients.

Cell type-specific knock out models to unravel NO/cGMP signaling in smooth muscle

NO-sensitive guanylyl cyclase (NO-GC) is accepted to be the major receptor for the signaling molecule NO. Deletion of NO-GC in mice leads to disturbed NO/cGMP signaling. As a result, these mice show abolished NO-dependent relaxation of smooth muscle-containing tissues in the cardiovascular and gastrointestinal systems. Mice with general deletion suffer from increased blood pressure, reduced bleeding time, erectile dysfunction and die prematurely due to gastrointestinal dysmotility. Several of the phenotypical changes caused by NO-GC deficiency are due to increased smooth muscle tone. Therefore, our work concentrated on NO/cGMP-mediated regulation of smooth muscle contraction/relaxation in various tissues. These include smooth muscle from blood vessels, gut, corpus cavernosum and lower urinary tract. NO-induced relaxation of all NO-GC-containing smooth muscle tissues was influenced by the deletion of the enzyme. To our surprise, the degree of smooth muscle dysfunction was not homogeneous: In contrast to vascular or urethral smooth muscle, gastric fundus and other GI muscles revealed an only a partially reduced NO-induced relaxation. Detrusor muscle of the bladder was unresponsive towards NO. Closer examination of the tissues showed variable expression of NO-GC in the different smooth muscle cells. Importantly, in addition to smooth muscle cells, many tissues showed NO-GC expression in other cell types such as endothelial cells, interstitial cells of Cajal and fibroblast-like cells. We have generated various cell-specific KO strains using the inducible Cre-lox-system. In the GI tract we were able to show a dual mechanism of NO-induced relaxation via interstitial cell of Cajal and smooth muscle cells. In addition, we identified a third type of NO-GC-expressing cell, the fibroblast-like cell, whose function is still enigmatic. In penile corpus cavernosum, strong NO-GC expression was found in smooth muscle cells and, surprisingly, also in endothelial cells. The function of NO-GC in the endothelium is currently being investigated. Further studies using double or triple KO mutants will hopefully allow advising cell-specific functions of NO/cGMP signaling in murine smooth muscle.

Red cell-derived microparticles (RMP) as haemostatic agent

Among circulating cell-derived microparticles, those derived from red cells (RMP) have been least well investigated. To exploit potential haemostatic benefit of RMP, we developed a method of producing them in quantity, and here report on their haemostatic properties. High-pressure extrusion of washed RBC was employed to generate RMP. RMP were identified and enumerated by flow cytometry. Their size distribution was assessed by Doppler electrophoretic light scattering analysis (DELSA). Interaction with platelets was studied by platelet aggregometry, and shear-dependent adhesion by Diamed IMPACT-R. Thrombin generation and tissue factor (TF) expression was also measured. The effect of RMP on blood samples of patients with bleeding disorders was investigated ex vivo by thromboelastography (TEG). Haemostatic efficacy in vivo was assessed by measuring reduction of blood loss and bleeding time in rats and rabbits. RMP have mean diameter of 0.45 µm and 50% of them exhibit annexin V binding, a proxy for procoagulant phospholipids (PL). No TF could be detected by flow cytometry. At saturating concentrations of MPs, RMP generated thrombin robustly but after longer delay compared to PMP and EMP. RMP enhanced platelet adhesion and aggregation induced by low-dose ADP or AA. In TEG study, RMP corrected or improved haemostatic defects in blood of patients with platelet and coagulation disorders. RMP reduced bleeding time and blood loss in thrombocytopenic rabbits (busulfan-treated) and in Plavix-treated rats. In conclusion, RMP has broad haemostatic activity, enhancing both primary (platelet) and secondary (coagulation) haemostasis, suggesting potential use as haemostatic agent for treatment of bleeding.

Prothrombin complex concentrates reduce blood loss in murine coagulopathy induced by warfarin, but not in that induced by dabigatran etexilate

Both established oral anticoagulants such as warfarin and newer agents such as dabigatran etexilate (DE) effectively prevent thromboembolic disease, but may provoke bleeding. Limited clinical data exist linking oral anticoagulant reversal and bleeding tendency, as opposed to surrogate laboratory markers. To quantify bleeding in warfarin-anticoagulated and DE-anticoagulated mice by tail transection with or without pretreatment with potential reversal agents: prothrombin complex concentrate (PCC); activated PCC (APCC); recombinant factor VIIa (rFVIIa); or murine fresh-frozen plasma (FFP). CD1 mice were given warfarin or DE by gavage, and the effects on in vitro coagulation assays, volume of blood loss and the bleeding time following tail transection injury were evaluated with different reversal agents. PCC (14.3 IU kg(-1) ), but not rFVIIa (3 mg kg(-1) ) or FFP (12 mL kg(-1) ), normalized blood loss and bleeding time in mice with warfarin-induced elevations of mean prothrombin time at two intensities (prothrombin time ratios of either 4.3 or 24). Neither separate nor combined PCC and/or rFVIIa treatment nor APCC (100 U kg(-1) ) treatment significantly reduced blood loss in mice anticoagulated with 60 mg kg(-1) DE 75 min prior to tail transection. Both combined PCC plus rFVIIa treatment and APCC treatment significantly reduced bleeding time in the DE-treated mice. Our data suggest that PCC treatment prevents excess bleeding much more effectively in warfarin-induced coagulopathy than in DE-induced coagulopathy.

In vivo and in vitro hemostatic activity of Chromolaena odorata leaf extract

Context: Chromolaena odorata (L.) R.M.King & H.Rob. (Asteraceae) or Siam weed has long been used to stop bleeding in Thailand and many countries. Only the aqueous leaf extract was investigated in in vivo and there have been conflicting results of in vitro hemostatic mechanisms of this plant.Objective: The most appropriate C. odorata leaf extract that promoted the highest hemostatic activity and the hemostatic mechanisms of these plant extracts will be investigated.Materials and methods: The lyophilized aqueous leaf extract and alcoholic (50, 70, and 95% ethanol) extracts from the fresh and dried leaves were investigated both in vivo and in vitro. The bleeding time in male Wistar rats was measured to investigate the hemostatic effect. The hemostatic mechanisms were tested using in vitro platelet aggregation and blood coagulation tests in sheep plasma.Results: All extracts displayed significantly reducing bleeding time (<2.5 min) in rats but did not induce platelet aggregation or blood clotting in the in vitro study. The in vitro blood clotting times of all extracts were > 0.6 min. Ethanol extract (70%) from the dried leaves proved to be the extract producing the highest hemostatic activity in vivo with the bleeding time of 1.85 min.Discussion and conclusion: The in vivo study with rats confirmed the significant ability of this plant extract to stop bleeding. However, the sufficient amount of calcium and active compounds which are aggregating and clotting agents to enhance blood coagulation and platelet aggregation in in vitro tests should be further studied.

Evaluation of the phytochemical and hemostatic potential of Jatropha multifida sap

Jatropha multifida is a plant traditionally recognized for its medicinal properties. This study was undertaken not only to assess the hemostatic potential of the sap of J. multifida, but also to elucidate its chemical profile. Wistar rats were injured at various locations and time of bleeding was evaluated based on the addition of J. multifida’s sap or distilled water. Phytochemical study was performed on sap. J. multifida’s sap significantly reduced bleeding time. Indeed, more blood flow was noticed on the side that received physiological water than the one which received the sap. This is even truer when the cut is deep with times of bleeding reduction of 39.06, 47.89, and 46.68%, respectively for superficial wounds, saphenous vein, and the femoral vein clippings. The phytochemical screening of J. multifida showed the presence of tannins, flavonoids, saponins, leucoanthocyanes, mucilage and reducing compounds. The power of the hemostatic sap is explained by the strong presence of tannins and flavonoids. All this proves that using the sap of J. multifida in traditional medicine is justified. Key words: Hemostatic, Wistar rats, tannins, flavonoids, bleeding, Jatropha multifida.

Accelerated hematopoietic recovery with angiotensin-(1–7) after total body radiation

Purpose: Angiotensin (1–7) [A(1–7)] is a component of the renin angiotensin system (RAS) that stimulates hematopoietic recovery after myelosuppression. In a Phase I/IIa clinical trial, thrombocytopenia after chemotherapy was reduced by A(1–7). In this study, the ability of A(1–7) to improve recovery after total body irradiation (TBI) is shown with specific attention to radiation-induced hematopoietic injury.Materials and methods: Mice were exposed to TBI (doses of 2–7 Gray [Gy]) of cesium 137 gamma rays, followed by treatment with A(1–7), typical doses were 100–1000 μg/kg given once or once daily for a specified number of days depending on the study. Animals are injected subcutaneously via the nape of the neck with 0.1 ml drug in saline. The recovery of blood and bone marrow cells was determined. Effects of TBI and A(1–7) on survival and bleeding time was also evaluated.Results: Daily administration of A(1–7) after radiation exposure improved survival (from 60% to 92–97%) and reduced bleeding time at day 30 after TBI. Further, A(1–7) increased early mixed progenitors (3- to 5-fold), megakaryocyte (2- to 3-fold), myeloid (3- to 6-fold) and erythroid (2- to 5-fold) progenitors in the bone marrow and reduced radiation-induced thrombocytopenia (RIT) (up to 2-fold). Reduction in the number of treatments to 3 per week also improved bone marrow recovery and reduced RIT. As emergency responder and healthcare systems in case of nuclear accident or/and terrorist attack may be overwhelmed, the consequence of delayed initiation of treatment was ascertained. Treatment with A(1–7) can be delayed up to 5 days and still be effective in the reduction of RIT or acceleration of bone marrow recovery.Conclusions: The data presented in this paper indicate that A(1–7) reduces the consequences of critical radiation exposure and can be initiated well after initial exposure with maximal effects on early responding hematopoietic progenitors when treatment is initiated 2 days after exposure and 5 days after exposure for the later responding progenitors and reduced thrombocytopenia. There was some effect of A(1–7) even when given days after radiation exposure.