Increased Bleeding Time Research Articles

Evaluation of Bleeding Time after Dental Extractions during Uninterrupted Single or Dual Antiplatelet Treatment - A Comparative Study

Abstract Introduction: This study aimed to assess the duration of bleeding after dental extractions amongst subjects with uninterrupted single antiplatelet therapy and dual antiplatelet therapy (DAPT) and to compare the bleeding time after dental extractions amongst those subjects undergoing various antiplatelet therapies. Materials and Methods: Post-extraction bleeding time was categorised as within 30 min, within 1 h and within 24 h. The bleeding time in different categories was compared and analysed using Chi-square. The antiplatelet agents assessed were aspirin, clopidogrel, ticagrelor and a combination of aspirin with clopidogrel and ticagrelor. Results: Bleeding time was significantly higher in patients under DAPT, compared to those under single antiplatelet therapy, and with an increase in the number of teeth extracted, there was an increase in bleeding time. All cases with prolonged bleeding could be managed with local haemostatic measures. Discussion: Simple extraction can be undertaken safely in patients under single antiplatelet therapy, considering that local haemostatic measures are available for use in the setup to control bleeding if necessary. Patients under DAPT are better managed if the therapy is altered, as there was a definite increase in bleeding time in patients under DAPT after extraction.

Antiplatelet and Antithrombotic Activities of Lespedeza cuneata via Pharmacological Inhibition of Integrin αIIbβ3, MAPK, and PI3K/AKT Pathways and FeCl3-Induced Murine Thrombosis.

Cardiovascular diseases (CVDs) have been the major cause of mortality all around the globe. Lespedeza cuneata abbreviated as L. cuneata with the authority name of Dumont de Courset (G. Don) is a perennial flowering plant commonly grown in Asian countries such as Korea, Japan, China, and Taiwan. We aimed to investigate the L. cuneata extract's antiplatelet and antithrombotic properties as GC-MS analysis indicated that the extract contained short-chain fatty acids, which have been reported to possess beneficial cardiovascular effects. L. cuneata was extracted using water, 50% EtOH, 70% EtOH, and 100% EtOH. For in vitro antiplatelet analysis, washed platelets were prepared and incubated with L. cuneata with 200 μg/mL of 50% EtOH in the presence of 1 mM of CaCl2 for 1 minute followed by agonist (collagen 2.5 μg/mL or ADP 10 μM or thrombin 0.1 U/mL) stimulation for 5 minutes over light transmission aggregometer. Scanning electron microscopy was performed to assess platelet shape change. ATP release and intracellular calcium mobilization were quantified to assess the granular content. Fibrinogen-binding assay and clot retraction assay assessed integrin αIIbβ3-mediated inside-out and outside-in signaling. Protein phosphorylation expression was investigated by western blot analysis. Finally, the in vivo antithrombotic efficacy was investigated by oral dosage of L. cuneata 200 and 400 mg/kg and aspirin 100 mg/kg for 7 days, and tail bleeding and FeCl3-induced murine thrombus model were performed. In vitro platelet aggregation and platelet shape change were dose-dependently suppressed by L. cuneata. Calcium mobilization, dense granules secretion, integrin αIIbβ3-mediated inside-out and outside-in signaling, and protein phosphorylation of MAPK and PI3K/Akt pathways were significantly inhibited. In vivo assays revealed that L. cuneata prevents side effects of synthetic drugs via nonsignificantly increasing bleeding time and improving coronary artery blood flow and animal survival. Our results demonstrate that L. cuneata exhibited potent antiplatelet and antithrombotic effects and can be considered a potential herbal medicine with cardioprotective effects.

HDAC Inhibitor CS014 Attenuates Thrombosis Alone and in Combination with Rivaroxaban without Increased Risk of Bleeding

Introduction: Targeting the human platelet for prevention of occlusive thrombotic events remains an active area of research due to the high incidence of myocardial infarction and stroke due to platelet-rich thrombotic clots. While, many patients taking FXa anticoagulant therapy require further inhibition of thrombosis, dual therapy including anticoagulant plus antiplatelet drugs result in an increased risk for bleeding. The current study sought to determine if a histone deacetylase (HDAC) inhibitor, either alone or in combination with the FXa inhibitor rivaroxaban, is able to effectively inhibit platelet and fibrin formation at the site of vascular injury without causing a significant increase in the bleeding risk in mice. Methods and Results: Thrombosis and bleeding models in the mouse were assessed in the presence of the HDAC inhibitors valproic acid (VPA) or the newly developed HDAC inhibitor, CS014. Both CS014 and VPA significantly, dose-dependently inhibit platelet accumulation and fibrin formation at the site of a vascular injury using the laser-induced cremaster thrombosis assay model. Further, to determine if CS014 has the potential to be used in conditions where an anticoagulant is already on board, mice were dosed for 5 days with rivaroxaban alone or in combination with CS014 and were assessed for prevention of laser-induced thrombosis in the cremaster arteriole. Both rivaroxaban and CS014 alone cause significant attenuation of clot formation. To determine if the combination results in an increased risk for bleeding, blood from mice treated with CS014 or rivaroxaban alone, or in combination was assessed for coagulation activity and clot strength using thromboelestography (TEG). Rivaroxaban was found to significantly delay onset of clotting in TEG whereas CS014 did not show any delay in coagulation or clotting with TEG. In combination CS014 was not observed to cause any additional delay in clotting time or decrease in maximal clot strength compared to rivaroxaban alone. Finally, a tail vein bleeding assay was conducted to determine if CS014 alone or in combination with rivaroxaban result in an increased risk for bleeding. Neither CS014 alone, rivaroxaban alone, or the combination of CS014 plus rivaroxaban resulted in increased bleeding time in the resected tail experiment. Conclusions: The data presented here suggests that while CS014 and rivaroxaban are each effective antithrombotic agents, they work well together to minimize clot formation without altering coagulation or clotting compared to rivaroxaban alone. Hence, CS014 has the potential to enrich the toolbox of anticoagulant therapies in patients with a high risk of thrombotic events.

Skrining Fitokimia dan Aktivitas Antikoagulan Sari Buah Melon (Cucumis melo L) Secara In Vivo

Anticoagulants are drugs used to inhibit the development and enlargement of a blood clot. Melon is known to contain adenosime that function to stop clotting of blood cell chip. This study aims to identify secondary metabolites contained in melon juice, fruit juice identify anticoagulant activity in vivo melon, look for an effective dose melon juice as a potential anticoagulant and melon juice compared with aspirin. A total of 15 male mice were divided into 5 groups, i.e. groups of melon juice dose of 12.9 g/kg BW, melon juice dose group of 25.8 g/kg BW, melon juice dose group of 51.5 g/kg BW, positive control group and negative control group. The sample is given once a day orally and measured in hours to 3, 6 and 9 after administration. Anticoagulant effect was tested by using the parameters of bleeding time and coagulation time. The results showed that the melon juice dose of 12.9 g/kg BW, 25.8 g/kg BW and 51.5 g/kg BW may increase bleeding time and coagulation time. From these results it can be seen that the melon juice has anticoagulant activity. Based on statistical test is known that the variation of doses is very significantly affect on bleeding time and the coagulation time.Keywords : anticoagulant, melon juice, bleeding time, coagulation time

Abstract 609: Heterozygous Tissue Factor Deficiency Protects Against Factor VII Activation And Consumption In A Murine Endotoxemia Model

Background: At least 1 in 25,000 humans carry F3 null alleles predicted to cause heterozygous tissue factor (TF) deficiency. Mice with heterozygous TF deficiency ( F3 +/- ) exhibit increased bleeding times and reduced thrombus formation in some models, and are protected against lethal thrombosis in penetrant thrombophilia. TF supports the activation of factor VII (FVII) in vitro , an effect that is evident in vivo in the extreme case of low-TF mice expressing ~1% of basal TF levels. Aim: To determine the impact of heterozygous TF deficiency on FVII activation in vivo . Methods: Citrated plasma was isolated via inferior vena cava blood draw from F3 +/- and wildtype littermate mice at 10-11 weeks of age. To induce coagulation activation through endotoxemia, mice were challenged with intraperitoneal injection of 8 mg/kg LPS for 6 hours. Plasma TF was determined by ELISA. FVII and FVIIa levels were measured by FVII activity and Staclot VIIa-rTF clotting assays, respectively. n≥10 mice in all experiments. Results: F3 +/- mice have significantly reduced plasma TF antigen levels compared to wildtype littermate controls under basal conditions (6.5 pg/mL vs 11.6 pg/mL, p<0.001), differences that persist following endotoxemic challenge (9.9 pg/mL vs 14.8 pg/mL, p<0.001). At baseline, F3 +/- mice had similar circulating levels of zymogen FVII and FVIIa compared to controls. However, the levels of FVIIa correlated with FVII activity levels in F3 +/- mice (r 2 =0.66, p=0.003), whereas no correlation between FVIIa and FVII activity levels was observed in controls (r 2 =0.17, p=0.23). During endotoxemia, F3 +/- mice maintained significantly higher total FVII (85.6% vs 66.1%, p<0.05) and FVIIa (52.9% vs 35.4%, p<0.05) as compared to controls. Conclusions: FVII and FVIIa levels are unaffected by heterozygous TF deficiency in healthy mice, but FVIIa levels are dependent on the amount of FVII when TF is limiting. When TF expression and coagulation activation are augmented as during endotoxemic challenge, F3 +/- mice are protected against aberrant FVII activation and consumption. These studies suggest that human genetic variation influencing TF dosage could influence FVII activation and disease outcomes.

Soluble endoglin reduces thrombus formation and platelet aggregation via interaction with αIIbβ3 integrin

BackgroundThe circulating form of human endoglin (sEng) is a cleavage product of membrane-bound endoglin present on endothelial cells. Because sEng encompasses an RGD motif involved in integrin binding, we hypothesized that sEng would be able to bind integrin αIIbβ3, thereby compromising platelet binding to fibrinogen and thrombus stability. MethodsIn vitro human platelet aggregation, thrombus retraction, and secretion-competition assays were performed in the presence of sEng. Surface plasmon resonance (SPR) binding and computational (docking) analyses were carried out to evaluate protein-protein interactions. A transgenic mouse overexpressing human sEng (hsEng+) was used to measure bleeding/rebleeding, prothrombin time (PT), blood stream, and embolus formation after FeCl3-induced injury of the carotid artery. ResultsUnder flow conditions, supplementation of human whole blood with sEng led to a smaller thrombus size. sEng inhibited platelet aggregation and thrombus retraction, interfering with fibrinogen binding, but did not affect platelet activation. SPR binding studies demonstrated that the specific interaction between αIIbβ3 and sEng and molecular modeling showed a good fitting between αIIbβ3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbβ3/sEng. hsEng+ mice showed increased bleeding time and number of rebleedings compared to wild-type mice. No differences in PT were denoted between genotypes. After FeCl3 injury, the number of released emboli in hsEng+ mice was higher and the occlusion was slower compared to controls. ConclusionsOur results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbβ3, suggesting its involvement in primary hemostasis control.

Chymase Inhibition Resolves and Prevents Deep Vein Thrombosis Without Increasing Bleeding Time in the Mouse Model.

Background Deep vein thrombosis (DVT) is the primary cause of pulmonary embolism and the third most life-threatening cardiovascular disease in North America. Post-DVT anticoagulants, such as warfarin, heparin, and direct oral anticoagulants, reduce the incidence of subsequent venous thrombi. However, all currently used anticoagulants affect bleeding time at various degrees, and there is therefore a need for improved therapeutic regimens in DVT. It has recently been shown that mast cells play a crucial role in a DVT murine model. The underlying mechanism involved in the prothrombotic properties of mast cells, however, has yet to be identified. Methods and Results C57BL/6 mice and mouse mast cell protease-4 (mMCP-4) genetically depleted mice (mMCP-4 knockout) were used in 2 mouse models of DVT, partial ligation (stenosis) and ferric chloride-endothelial injury model of the inferior vena cava. Thrombus formation and impact of genetically repressed or pharmacologically (specific inhibitor TY-51469) inhibited mMCP-4 were evaluated by morphometric measurements of thrombi immunochemistry (mouse and human DVT), color Doppler ultrasound, bleeding times, and enzymatic activity assays ex vivo. Recombinant chymases, mMCP-4 (mouse) and CMA-1 (human), were used to characterize the interaction with murine and human plasmin, respectively, by mass spectrometry and enzymatic activity assays. Inhibiting mast cell-generated mMCP-4, genetically or pharmacologically, resolves and prevents venous thrombus formation in both DVT models. Inferior vena cava blood flow obstruction was observed in the stenosis model after 6 hours of ligation, in control- but not in TY-51469-treated mice. In addition, chymase inhibition had no impact on bleeding times of healthy or DVT mice. Furthermore, endogenous chymase limits plasmin activity in thrombi ex vivo. Recombinant mouse or human chymase degrades/inactivates purified plasmin in vitro. Finally, mast cell-containing immunoreactive chymase was identified in human DVT. Conclusions This study identified a major role for mMCP-4, a granule-localized protease of chymase type, in DVT formation. These findings support a novel pharmacological strategy to resolve or prevent DVT without affecting the coagulation cascade through the inhibition of chymase activity.

Antithrombotic and Antioxidant Activities of Binahong [Anredera cordifolia (Ten.) Steenis] Leaf Ethanol Extract and Its Nanoemulsion Preparation in Swiss Webster Mice

Platelet aggregation has the potential to form thrombi and result in cardiovascular system diseases such as myocardial infarction and ischemic stroke - one of the leading causes of death globally. Traditionally, people use binahong leaves as blood thinners. Therefore, this study aims to obtain scientific data on the efficacy of binahong leaf as antithrombotic. The efficacy test was carried out on male Swiss-Webster mice. The results showed that the ethanol extract of binahong leaves at doses of 50 (BLEE50) and 100 mg/kg bw (BLEE100) could increase bleeding time on H7 (7.61±1.79% and 3.72±1.76% vs 1.08±0.90%) and H14 (13.81±4.42% and 5.06±2.30% vs 1.66±1.09%) and coagulation time at H7 (5.01±1.36% and 4.18±1.67% vs 1.38±1.08%) and at H14 (7.92±1.97% and 7.19±1.96% vs 1.70±1.10%) significantly (p<0.05). The two doses of BLEE were formulated in the form of nanoemulsions with the Self Nano Emulsifying Drug Delivery System (NBLEE50 and NBLEE100) were also able to prolong bleeding time and coagulation time significantly (p<0.01) but only NBLEE50 prolonged bleeding time (p<0.05) significantly against BLEE50. In the test of the anti-platelet aggregation effect with ADP as an inducer, both doses of BLEE and the nanoemulsion preparation (NBLEE) could significantly (p<0.01) inhibit platelet aggregation with a percentage of inhibition >70% which was not different from the standard (acetylsalicylic acid). In the antioxidant effect test using the DPPH method, BLEE has an IC50 = 66.08?g/mL which is classified as a strong antioxidant. Both doses of BLEE and its NBLEE could significantly (p<0.01) inhibit lipid peroxidation in plasma and liver and NO radicals formation. BLEE50 can significantly (p<0.05) reduce mean platelet volume (6.05±0.24fL vs 6.55±0.34fL) and platelet distribution width (8.52±0.36% vs 9.25±0.42%). Based on those results, BLEE has the potential to be used as an antiplatelet aggregation and antioxidant.

Abstract 12521: TMEM16 Antagonists Inhibit Phosphatidylserine-Induced Procoagulant Activity in Endothelial Cells and Protect From Thrombosis in vivo

Introduction: Constitutively an anticoagulant surface, endothelial cells (ECs) switch to support coagulation following pathogenic stimuli, contributing to cardiovascular disease. To promote thrombosis, coagulation proteins must assemble on a membrane surface containing the anionic phospholipid phosphatidylserine (PS). PS is asymmetrically distributed on the inner leaflet of the plasma membrane but is rapidly externalized to the outer leaflet by calcium-activated phospholipid “scramblases”, such as TMEM16F. How phospholipid externalization on ECs supports coagulation and thrombosis has not been investigated. Hypothesis: We proposed that pharmacologic inhibition of TMEM16 phospholipid scramblases inhibits EC procoagulant activity and protects against thrombosis. Methods and Results: siRNA silencing of TMEM16F, and its closest paralog, TMEM16E, inhibited factor Xa generation and thrombin generation on the EC surface (60% and 90%, respectively, p < 0.0001), equivalent to the PS-binding protein lactadherin (100 nM). Two unrelated compounds with TMEM16 inhibitory activity, benzbromarone and CaCCinh-A01 (30 μM), prevented calcium-induced PS externalization on ECs, as determined by flow cytometry and immunofluorescence microscopy. These drugs did not inhibit intracellular calcium flux, suggesting they block the PS externalization activity of TMEM16. Benzbromarone and CaCCinh-A01 inhibited factor Xa generation on the EC surface in a dose-dependent manner (IC 50 3.2 μM and 2.0 μM, respectively). Thrombosis was evaluated using a laser injury model in the mouse cremasteric arteriole coupled with intravital microscopy to measure accumulation of fluorescently-labeled platelets and fibrin. Benzbromarone (5 mg/kg) inhibited platelet accumulation ( p < 0.05) and fibrin formation ( p < 0.01). Benzbromarone did not increase bleeding time in a tail-clip hemostasis assay. Conclusions: Activated ECs contribute anionic phospholipid to support coagulation, a process regulated by TMEM16 phospholipid scramblases. TMEM16 inhibition with benzbromarone protects from experimental thrombosis without resulting in excess bleeding. Benzbromarone is used worldwide to treat gout and could be repurposed for anti-thrombotic properties.

A PROSPECTIVE STUDY ON EFFECT OF FLUOXETINE ON PRIMARY HEMOSTASIS OF PATIENTS HAVING MAJOR DEPRESSIVE DISORDER

Objectives: The objectives of the study were to study the effect of fluoxetine on bleeding time, clotting time and platelet count of depressed patients. Methods: Patients diagnosed with major depressive disorder were included in the study to fulfill a sample size of 60. Before starting the treatment with fluoxetine, laboratory tests were done which included bleeding time, clotting time, and platelet count. Patients were requested to return for follow-up after 4 weeks of treatment and the laboratory tests were repeated. All the study end point analysis was analyzed based on per-protocol population. Continuous variables were expressed as mean and standard deviations, paired t-test was used for within group comparison and unpaired t-test was used for between group comparisons. p<0.05 was considered to be significant. For categorical variable, frequency and percentage were calculated. For continuous variable, that is, bleeding time, clotting time, and platelet count, mean and standard deviation were calculated. Results: At the end of 4 weeks, it was observed that there was a significant increase in bleeding time from 1.35±0.08 min to 1.46±0.08 min**. Similarly, there was a significant increase in clotting time from 3.30±0.15 min to 3.38±0.15 min**. It was also observed that there was a significant decrease in platelet count from 3.07±0.67 lakh cells/cu mm to 2.86±0.63 lakh cells/cu mm**. Conclusion: Fluoxetine has shown to increase bleeding time, clotting time, and decrease platelet count. Hence, fluoxetine induced risk of bleeding and its cardio protective action has to be considered while individualizing therapy in management of depression.

Clopidogrel delays and can reverse diabetic nephropathy pathogenesis in type 2 diabetic db/db mice.

BACKGROUNDDiabetic nephropathy (DN) is the principal cause of end-stage renal disease. Previous studies have shown that clopidogrel can prevent the early progression of renal injury.AIMTo elucidate whether clopidogrel is beneficial against DN by using a db/db mouse model.METHODS db/db mice with a higher urinary albumin/creatinine ratio (ACR) relative to age- and sex-matched wild-type control mice were randomly allocated to clopidogrel and vehicle treatment groups. Clopidogrel was administered at doses of 5, 10, and 20 mg/kg by gavage for 12 wk. Body mass, blood glucose level, and urinary creatinine and albumin concentrations in each group were measured before and after the intervention. Renal fibrosis was evaluated using periodic acid-Schiff and Masson’s trichrome staining. The renal protein expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and F4/80 was assessed using immunohistochemistry. Urinary TNF-α, monocyte chemoattractant protein-1 (MCP-1), and IL-6 levels were analyzed using enzyme-linked immunosorbent assay; TNF-α and IL-1β mRNA expression was measured using real-time quantitative polymerase chain reaction. The protein expression of fibronectin (FN) and collagen I was assessed using immunohistochemistry.RESULTSClopidogrel treatment did not affect the body mass or blood glucose level of the db/db mice; however, it increased bleeding time and reduced urinary ACR in a dose-dependent manner. Immunohistochemical staining revealed an amelioration of renal fibrosis, significantly lower deposition of FN and collagen I, and significantly lower expression of the proinflammatory cytokines TNF-α and IL-1β and lower levels of urinary TNF-α and MCP-1 in the clopidogrel-treated db/db mice (P < 0.05). Furthermore, clopidogrel significantly reduced macrophage infiltration into the glomeruli of the db/db mice.CONCLUSIONClopidogrel significantly reduced renal collagen deposition and fibrosis and prevented renal dysfunction in db/db mice, most likely through inhibition of renal macrophage infiltration and the associated inflammation.

Pharmacological profile of asundexian, a novel, orally bioavailable inhibitor of factor XIa

BackgroundActivated coagulation factor XI (FXIa) contributes to the development and propagation of thrombosis but plays only a minor role in hemostasis; therefore, it is an attractive antithrombotic target. ObjectivesTo evaluate the pharmacology of asundexian (BAY 2433334), a small molecule inhibitor targeting FXIa, in vitro and in various rabbit models. MethodsThe effects of asundexian on FXIa activity, selectivity versus other proteases, plasma thrombin generation, and clotting assays were evaluated. Antithrombotic effects were determined in FeCl2‐ and arterio‐venous (AV) shunt models. Asundexian was administered intravenously or orally, before or during thrombus formation, and with or without antiplatelet drugs (aspirin and ticagrelor). Potential effects of asundexian on bleeding were evaluated in ear‐, gum‐, and liver injury models. ResultsAsundexian inhibited human FXIa with high potency and selectivity. It reduced FXIa activity, thrombin generation triggered by contact activation or low concentrations of tissue factor, and prolonged activated partial thromboplastin time in human, rabbit, and various other species, but not in rodents. In the FeCl2‐injury models, asundexian reduced thrombus weight versus control, and in the arterial model when added to aspirin and ticagrelor. In the AV shunt model, asundexian reduced thrombus weight when administered before or during thrombus formation. Asundexian alone or in combination with antiplatelet drugs did not increase bleeding times or blood loss in any of the models studied. ConclusionsAsundexian is a potent oral FXIa inhibitor with antithrombotic efficacy in arterial and venous thrombosis models in prevention and intervention settings, without increasing bleeding.

Ebola Survivors are not at Increased Risk for Gynecologic Surgeries

As the result of multiple signs and symptoms, and complications observed among survivors of the Ebola virus disease (EVD), there is an assumption that survivors might experience perturbations within their clotting parameters. This may eventually lead to increased bleeding time, predisposing them to increased risk for surgical complications. This study aimed to comparatively review specific intra-operative parameters such as uterine fibroids and polyps among a number of EVD survivors and non survivors undergoing elective gynecologic surgeries at Redemption Hospital, a tertiary specialized referral hospital located within one of the EVD hotspots, in Liberia from January to October 2016. A case-control study was conducted wherein cases were referred from the Partnership for = Research on Vaccines and Infectious Diseases in Liberia (PREVAIL), an EVD clinical trial platform, while controls were sampled from the general patient population, at Redemption Hospital. The controls were matched based on age, employment status and parity. All surgeries were performed by single surgeon based on a surgical checklist that included designated intra- and post-operative parameters. Statistical analysis such as counts, percentiles, confidence intervals and relative risks were performed to assess the differences between the cases and controls, respectively. Survivors were between the ages of 42 years and 44 years for controls with an average interval between discharge from the Ebola Treatment Unit (ETU) and surgical intervention of greater than one year. The median duration of surgical procedure was 60 minutes and blood loss of 250 ml in both groups. Besides the relative risks (RR) of receiving antibiotics for more than 3 days of 1.5 (85.7% vs. 57.1%) and hospitalization of more than 7 days of 0.25 (14% vs. 57%) for survivors as compared to controls, most of the indicators were not significantly different. The findings revealed that EVD survivors who present with benign tumors such as uterine fibroids, polyps, or adenomyosis, requiring elective uterine surgery such as myomectomy and/or hysterectomy are generally not at increased risk of surgical complications because most of the indicators (hospitalization, blood loss, antibiotics, etc.) were not significantly different between the two groups. Findings from this study may potentially revise the approaches used by gynecologists and general surgeons during encounters and/or interventions with patient(s) concerning emerging infectious diseases (EIDs)

A STUDY OF PLATELET COUNT AND BLEEDING TIME IN THIRD TRIMESTER OF PREGNANCY WITH REFERENCE TO PRE-ECLAMPSIA.

BACKGROUND: - Pre-eclampsia is a serious complication of pregnancy and contributes signicantly maternal as well as perinatal morbidity and mortality. So an attempt was made to study the platelet count and bleeding time in cases of preeclampsia. AIM:-(1) To nd out the platelet count and bleeding time in pre-eclamptic woman. rd (2) To compare the values of platelet count and bleeding time in pre-eclamptics and normal pregnant woman in 3 trimester of pregnancy. METHODOLOGY:- 120 pregnant women in age group 18-42 years were included in the study of which 40 women were mild pre-eclamptic, 40 severe pre-eclamptic and 40 normal pregnant women in third trimester of pregnancy. RESULT:- Signicant decrease in platelet count and signicant increase in bleeding time was found between pre-eclamptic in comparison with the normal pregnant women. DISCUSSION:- Platelet count was found to decrease &amp; Bleeding Time increase in pre-eclampsia CONCLUSION:- From this study,it was concluded that platelet count decrease and bleeding time incease with increase in severity of preeclampsia

Discovery of Milvexian, a High-Affinity, Orally Bioavailable Inhibitor of Factor XIa in Clinical Studies for Antithrombotic Therapy.

Factor XIa (FXIa) is an enzyme in the coagulation cascade thought to amplify thrombin generation but has a limited role in hemostasis. From preclinical models and human genetics, an inhibitor of FXIa has the potential to be an antithrombotic agent with superior efficacy and safety. Reversible and irreversible inhibitors of FXIa have demonstrated excellent antithrombotic efficacy without increased bleeding time in animal models (Weitz, J. I., Chan, N. C. Arterioscler. Thromb. Vasc. Biol. 2019, 39 (1), 7-12). Herein, we report the discovery of a novel series of macrocyclic FXIa inhibitors containing a pyrazole P2' moiety. Optimization of the series for (pharmacokinetic) PK properties, free fraction, and solubility resulted in the identification of milvexian (BMS-986177/JNJ-70033093, 17, FXIa Ki = 0.11 nM) as a clinical candidate for the prevention and treatment of thromboembolic disorders, suitable for oral administration.

CIB1 Regulates Hemostasis through Integrin Outside-in Signaling

On circulating platelets, integrin αIIbβ3, the fibrinogen receptor, is in a low affinity state unable to bind soluble ligands. Agonist-dependent inside-out signaling is necessary for activation of the integrin αIIbβ3 in order to bind ligands such as fibrinogen (Fg). Ligand binding induces a cascade of signaling that culminates in platelet aggregation, leading to hemostatic plug formation and clot retraction. Although it is well known that signaling through integrin αIIbβ3 is necessary, the mechanism of clot retraction is not well understood. Previously, we have identified a calcium- and integrin-binding protein 1 (CIB1) that specifically interacts with integrin αIIbβ3. One study implicated CIB1 as an endogenous inhibitor of integrin αIIbβ3 activation, while another study suggested CIB1 to be an activator of inside-out signaling. Using a novel technique to inhibit interaction of CIB1 with integrin in vivo, we have shown that CIB1 regulates outside-in signaling through integrin αIIbβ3. Definitive proof for the role of CIB1 in platelets remained absent, however. Here, using Cib1 null platelets, we show for the first time that CIB1 is a key regulator of hemostasis. Genetic ablation of CIB1 had no effect on agonist-induced platelet aggregation, excluding the role for CIB1 in inside-out signaling. However, deletion of the Cib1 gene significantly increased mouse bleeding times. More than 50% of the mice also showed a rebleeding phenotype, suggesting a defect in clot stability. Incomplete and significantly delayed clot retraction was observed in Cib1 null platelets compared to wildtype littermates. The absence of CIB1 also affects the ability of platelets to spread on immobilized Fg. Platelet adhesion and subsequent filopodia formation was not affected by the absence of CIB1. Platelet spreading can be restored by addition of exogenous ADP, suggesting that the defect is not in the ability of the platelet to spread. These in vivo and in vitro results clearly show that CIB1 regulates hemostasis by supporting transition of platelets from filopodial stage to fully spread morphology during outside-in signaling through integrin αIIbβ3.

Platelet Dysfunction and Thrombosis in JAK2V617F-Mutated Primary Myelofibrotic Mice.

The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2V617F (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2V617F transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl3-induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation. Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.

Study of haematological and coagulation profile of Malaria in Tertiary level care centre in Ghaziabad

Objective: To study some hematological parameters in patients with complicated severe malaria and their relations to clinical outcome. Materials and Methods: This was a cross sectional observational study, including 100 patients from a tertiary care centre in Ghaziabad patients who were diagnosed to have malaria by peripheral smear examination (both thick and thin smear) or Malaria Rapid Antigen. Blood sample for haematological and coagulation study were taken. Results: Out of 100 patients 62 patients had Hb % less than 10 gm percentage in which 14 patients had Hb < 6> of the total patients. Packed cell volume was decreased in about 86% of the patients. ESR increase was seen in 73% of the patient. Thrombocytopenia was observed in 63% of the patients and 25% of the patients with mixed infection had severe thrombocytopenia. PT and APTT was increased in 22% and 14% of the total cases. Bleeding time was increased in 5% of the total case and no any patients with vivax malaria had increased bleeding time. Mixed infection shows as falciparum malaria. Sever anemia, thrombocytopenia, increase BT, PT and APTT associated to severity and complication of malaria. Conclusion: This study concluded that hematological changes are common complications encountered in severe malaria, but they are not related to the clinical outcome. Keywords: Severe malaria, Thrombocytopenia, Prothrombin time, APTT.

The choline transporter Slc44a2 controls platelet activation and thrombosis by regulating mitochondrial function

Genetic factors contribute to the risk of thrombotic diseases. Recent genome wide association studies have identified genetic loci including SLC44A2 which may regulate thrombosis. Here we show that Slc44a2 controls platelet activation and thrombosis by regulating mitochondrial energetics. We find that Slc44a2 null mice (Slc44a2(KO)) have increased bleeding times and delayed thrombosis compared to wild-type (Slc44a2(WT)) controls. Platelets from Slc44a2(KO) mice have impaired activation in response to thrombin. We discover that Slc44a2 mediates choline transport into mitochondria, where choline metabolism leads to an increase in mitochondrial oxygen consumption and ATP production. Platelets lacking Slc44a2 contain less ATP at rest, release less ATP when activated, and have an activation defect that can be rescued by exogenous ADP. Taken together, our data suggest that mitochondria require choline for maximum function, demonstrate the importance of mitochondrial metabolism to platelet activation, and reveal a mechanism by which Slc44a2 influences thrombosis.

Ulmus parvifolia Modulates Platelet Functions and Inhibits Thrombus Formation by Regulating Integrin αIIbβ3 and cAMP Signaling.

BackgroundThe prevalence of cardiovascular diseases (CVDs) is increasing at a high rate, and the available treatment options, sometimes, have complications which necessitates the need to develop safer and efficacious approaches. Ethnomedicinal applications reportedly reduce CVD risk. Ulmus parvifolia Jacq. (Ulmaceae) commonly known as Chinese Elm or Lacebark Elm, is native to China, Japan, and Korea. It exhibits anti-inflammatory, antiviral, and anticancer properties, but its anti-platelet properties have not yet been elucidated.PurposeTo investigate the pharmacological anti-platelet and anti-thrombotic effects of U. parvifolia bark extract.Study Design and MethodsHuman and rat washed platelets were prepared; light transmission aggregometry and scanning electron microscopy was performed to assess platelet aggregation and the change in platelet shape, respectively. Intracellular calcium mobilization, ATP release, and thromboxane-B2 production were also measured. Integrin αIIbβ3 activation was analyzed in terms of fibrinogen binding, fibronectin adhesion, and clot retraction. The expression of MAPK, Src, and PI3K/Akt pathway proteins was examined. Cyclic nucleotide signaling pathway was evaluated via cAMP elevation and VASP phosphorylation. Anti-thrombotic activity of the extract was evaluated in vivo using an arteriovenous shunt rat model, whereas its effect on hemostasis in mice was assessed via bleeding time assay.Results U. parvifolia extract significantly inhibited human and rat platelet aggregation in a dose-dependent manner along with inhibition of calcium mobilization, dense granule secretion, and TxB2 production. Integrin αIIbβ3 mediated inside-out and outside-in signaling events, as evidenced by the inhibition of fibrinogen binding, fibronectin adhesion, and clot retraction. The extract significantly reduced phosphorylation of Src, MAPK (ERK, JNK, and p38MAPK), and PI3K/Akt pathway proteins. Cyclic-AMP levels were elevated in U. parvifolia-treated platelets, while PKAαβγ and VASPser157 phosphorylation was enhanced. U. parvifolia reduced thrombus weight in rats and moderately increased bleeding time in mice.Conclusion U. parvifolia modulates platelet responses and inhibit thrombus formation by regulating integrin αIIbβ3 mediated inside-out and outside-in signaling events and cAMP signaling pathway.