N-Acetyl Cysteine Prevents Arterial Thrombosis in a Dose-Dependent Manner In Vitro and in Mice.

BACKGROUND: Platelet-rich thrombi occlude arteries causing fatal infarcts like heart attacks and strokes. Prevention of thrombi by current antiplatelet agents can cause major bleeding. Instead, we propose using N-acetyl cysteine (NAC) to act against the protein VWF (von Willebrand factor), and not platelets, to prevent arterial thrombi from forming. METHODS: NAC was assessed for its ability to prevent arterial thrombosis by measuring platelet accumulation rate and occlusion time using a microfluidic model of arterial thrombosis with human blood. Acute clot formation, clot stability, and tail bleeding were measured in vivo with the murine modified Folts model. The effect of NAC in the murine model after 6 hours was also measured to determine any persistent effects of NAC after it has been cleared from the blood. RESULTS: We demonstrate reduction of thrombi formation following treatment with NAC in vitro and in vivo. Human whole blood treated with 3 or 5 mmol/L NAC showed delayed thrombus formation 2.0× and 3.7× longer than control, respectively (P<0.001). Blood treated with 10 mmol/L NAC did not form an occlusive clot, and no macroscopic platelet aggregation was visible (P<0.001). In vivo, a 400-mg/kg dose of NAC prevented occlusive clots from forming in mice without significantly affecting tail bleeding times. A lower dose of NAC significantly reduced clot stability. Mice given multiple injections showed that NAC has a lasting and cumulative effect on clot stability, even after being cleared from the blood (P<0.001). CONCLUSIONS: Both preclinical models demonstrate that NAC prevents thrombus formation in a dose-dependent manner without significantly affecting bleeding time. This work highlights a new pathway for preventing arterial thrombosis, different from antiplatelet agents, using an amino acid derivative as an antithrombotic therapeutic.

Relationships of apolipoprotein E genotypes with a cluster of seven in persons with type 2 diabetes.

OBJECTIVE.: The objective of the study was to determine if there would be statistically significant differences or trends among apolipoprotein E genotypes (2/2, 2/3, 2/4, 3/3, 3/4, and 4/4) for each member of the cluster of seven associated with type 2 diabetes (T2D). The cluster of seven includes abdominal obesity, hypertension, platelet hyperaggregability, hyperglycemia, dyslipidemia (decreased plasma levels of high-density lipoprotein cholesterol (HDL-C) and increased plasma levels of triglycerides)), increased low-density lipoprotein (LDL) oxidation, and increased inflammation. METHODS.: Forty-six patients with well-controlled T2D participated in the study. Abdominal obesity (assessed by waist circumference), hypertension (measured by manual sphygmomanometry), platelet hyperaggregability (measured by bleeding time), hyperglycemia (by enzymatic kit and spectrophotometry), decreased plasma levels of HDL-C and increased plasma levels of triglycerides (by enzymatic kit and spectrophotometry), increased LDL oxidation (measured by LDL conjugated dienes using spectrophotometry) and increased inflammation measured by C-reactive protein (CRP) (by EIA kit) were determined. RESULTS.: All genotypes, except 2/2 were found in the population studied. Abdominal obesity did not vary significantly across the five genotypes. However, glucose levels trended progressively higher going from 2/3 to 2/4 to 3/4 to 4/4. Systolic blood pressure was higher in 3/4 compared to 2/4 and trended higher in 3/4 compared to 3/3. Diastolic blood pressure trended higher in 3/3 vs 2/4 and significantly higher in 3/4 compared to 2/4. Triglycerides trended higher in 3/4 vs 3/3 while HDL-C came close to trending downward in 4/4 compared to 2/4. Bleeding time was unaffected by genotype. Plasma LDL conjugated dienes trended higher in 3/4 vs 2/4 and were significantly higher in 3/4 vs 3/3. CRP trended higher in 4/4 vs 2/3. CONCLUSION.: We can conclude that those with at least one 4 allele in the presence of another allele being 2, 3 or 4 is potentially (in the case of trends) deleterious or is deleterious in terms of hyperglycemia, hypertension (systolic and diastolic blood pressure), dyslipidemia, LDL conjugated dienes and CRP levels.

Comparison of laser and traditional lancing devices for capillary blood sampling in patients with diabetes mellitus and high bleeding risk.

PURPOSE: Despite the importance of self-monitoring blood glucose (SMBG) for management of diabetes mellitus (DM), frequent blood sampling is discouraged by bleeding risk due to dual-antiplatelet agent therapy (DAPT) or thrombocytopenia. METHODS: We compared the bleeding time (BT) of sampling by using a laser-lancing-device (LMT-1000) and a conventional lancet in patients with DM and thrombocytopenia or patients undergoing DAPT. BT was measured using the Duke method, and pain and satisfaction scores were assessed using numeric rating scale (NRS) and visual analog scale (VAS). The consistency in the values of glucose and glycated-hemoglobin (HbA1c) sampled using the LMT-1000 or lancet were compared. RESULTS: The BT of sampling with the LMT-1000 was shorter than that with the lancet in patients with thrombocytopenia (60s vs. 85s, P = 0.024). The NRS was lower and the VAS was higher in laser-applied-sampling than lancet-applied sampling in the DAPT-user group (NRS: 1 vs. 2, P = 0.010; VAS: 7 vs. 6, P = 0.003), whereas the group with thrombocytopenia only showed improvement in the VAS score (8 vs. 7, P = 0.049). Glucose and HbA1c sampled by the LMT-1000 and lancet were significantly correlated in both the DAPT-user and the thrombocytopenia groups. CONCLUSION: The LMT-1000 can promote SMBG by shortening BT in subject with thrombocytopenia and by increasing satisfaction score, as well as by showing reliable glucose and HbA1c value.

Over-expression of Dyrk1A affects bleeding by modulating plasma fibronectin and fibrinogen level in mice.

Down syndrome is the most common chromosomal abnormality in humans. Patients with Down syndrome have hematologic disorders, including mild to moderate thrombocytopenia. In case of Down syndrome, thrombocytopenia is not associated with bleeding, and it remains poorly characterized regarding molecular mechanisms. We investigated the effects of overexpression of Dyrk1A, an important factor contributing to some major Down syndrome phenotypes, on platelet number and bleeding in mice. Mice overexpressing Dyrk1A have a decrease in platelet number by 20%. However, bleeding time was found to be reduced by 50%. The thrombocytopenia and the decreased bleeding time observed were not associated to an abnormal platelet receptors expression, to a defect of platelet activation by ADP, thrombin or convulxin, to the presence of activated platelets in the circulation or to an abnormal half-life of the platelets. To propose molecular mechanisms explaining this discrepancy, we performed a network analysis of Dyrk1A interactome and demonstrated that Dyrk1A, fibronectin and fibrinogen interact indirectly through two distinct clusters of proteins. Moreover, in mice overexpressing Dyrk1A, increased plasma fibronectin and fibrinogen levels were found, linked to an increase of the hepatic fibrinogen production. Our results indicate that overexpression of Dyrk1A in mice induces decreased bleeding consistent with increased plasma fibronectin and fibrinogen levels, revealing a new role of Dyrk1A depending on its indirect interaction with these two proteins.

Circadian rhythm and aneurysmal subarachnoid hemorrhage: Is there an alarm clock for the rupture timing?

BACKGROUND AND PURPOSE: Data on the temporal distribution of the bleeding time of intracranial aneurysms are limited to a few small studies. With this study, the aim was to analyze time patterns of the occurrence of aneurysmal subarachnoid hemorrhage (SAH), particularly focusing on the impact of patients' socio-demographic and clinical characteristics on the ictus timing. METHODS: The study is based on an institutional SAH cohort with 782 consecutive cases treated between January 2003 and June 2016. Data were collected on the ictus time, patients' socio-demographic and clinical characteristics, as well as the initial severity and outcome. Univariate and multivariate analyses were performed on the bleeding timeline. RESULTS: There were two peaks in the circadian rhythm of SAH, one in the morning (7-9 a.m.) and the other in the evening (7-9 p.m.). The strongest alterations in the bleeding time patterns were observed for weekdays, patients' age, sex and ethnicity. Individuals with chronic alcohol and painkiller consumption showed a higher bleeding peak between 1 and 3 p.m. Finally, the bleeding time showed no impact on the severity, clinically relevant complications and the outcome of SAH patients. CONCLUSIONS: This study is one of the very few detailed analyses of the impact of specific socio-demographic, ethnic, behavioral and clinical characteristics on the rupture timing of aneurysms. Our results point to the possible relevance of the circadian rhythm for the rupture event, and therefore might be useful in the elaboration of preventive measures against aneurysm rupture.

Towards 50 years of platelet function analyser (PFA) testing.

The platelet function analyser (PFA) is a prevalent platelet function screening instrument, and comes in two models-the original PFA-100 and the contemporary PFA-200. The instruments have 'identical' output, being a 'closure time' (CT). Moreover, normal reference ranges provided by the manufacturer, for the specific test cartridges, are the same for both models. There are three different types of test cartridge: collagen/epinephrine (C/Epi), collagen/adenosine diphosphate (C/ADP), and "Innovance PFA P2Y" (only available in certain geographical locations). The PFA-100 was released in the mid 1990s, and so is approaching 50 years of age. The PFA-200, released in some locations in the mid 2010s, is destined to eventually replace the PFA-100, but is not yet available in the USA. The test system is highly sensitive to von Willebrand disease (VWD; C/Epi and C/ADP) and to aspirin therapy (C/Epi only), but only has moderate sensitivity to defects in platelet function and/or deficiencies in platelet number. Accordingly, recommendations for use for screening platelet function vary according to user experience. Some workers have alternatively used the PFA to assess thrombosis risk or pre-operative bleeding risk. In this review, we provide an overview of the history of PFA, and summarise its current clinical utility.

Application of Design of Experiment in the Optimization of Apixaban-Loaded Solid Lipid Nanoparticles: In Vitro and In Vivo Evaluation.

Solid lipid nanoparticles (SLnPs) are usually utilized as lipid-based formulations for enhancing oral bioavailability of BCS class IV drugs. Accordingly, the objective of this work was to investigate the effect of formulation and processing variables on the properties of the developed SLnPs for oral delivery of apixaban. Randomized full factorial design (24) was employed for optimization of SLnPs. With two levels for each independent variable, four factors comprising both formulations and processing factors were chosen: the GMS content (A), the Tween 80 content (B), the homogenization time (C), and the content of poloxamer 188 used (D). The modified hot homogenization and sonication method was employed in the formulation of solid lipid nanoparticles loaded with apixaban (APX-SLnPs). The size of APX-SLnPs formulations was measured to lie between 116.7 and 1866 nm, polydispersity index ranged from 0.385 to 1, and zeta potential was discovered to be in the range of - 12.6 to - 38.6 mV. The entrapping efficiency of APX-SLnPs formulations was found to be in the range of 22.8 to 96.7%. The optimized formulation was evaluated in vivo after oral administration to rats. Oral administration of APX-SLnPs resulted in significant prolongation in bleeding time compared with both positive and negative control. This indicates the ability of this system to enhance drug therapeutic effect either by increasing intestinal absorption or trans-lymphatic transport. So, this study highlighted the capability of SLnPs to boost the pharmacological effect of apixaban.

Hemostatic function of cold-stored platelets in a thrombocytopenic rabbit bleeding model.

BACKGROUND: Transfusion of cold-stored platelet concentrates (CS-PCs) appears effective in massively bleeding patients. However, few studies have evaluated their in vivo hemostatic function in severe thrombocytopenia. STUDY DESIGN AND METHODS: The in vivo function of plasma-depleted human PCs was evaluated in rabbits with a blocked reticuloendothelial system and busulfan-induced thrombocytopenia. On day 1, a human apheresis PC was processed in a platelet additive solution (PAS-PC) and split evenly for cold or room temperature storage (RTS). On days 3, 6, or 9, RTS- or CS-PAS-PCs were transfused (4.0 × 109 platelets/kg) after plasma depletion into two to four rabbits that developed adequate thrombocytopenia (<25 × 109 /L). Ear bleeding time was measured by two incisions in small veins. The hemostatic rate was defined as the percentage of rabbits achieving bleeding cessation within 600 s at either incision. The experiment was repeated using five different PCs on each storage day. RESULTS: The mean pre-transfusion rabbit platelet count was 8.6 ± 5.2 × 109 /L. The hemostatic rates with RTS- and CS-PAS-PCs were both 100% on day 3, 93 ± 15% and 73 ± 15% on day 6 (p = .07), and 65 ± 36% and 73 ± 37% on day 9 (p = .27), respectively, with no statistical differences. Total platelet counts were significantly lower after CS-PAS-PC than RTS-PAS-PC transfusion on all days (e.g., 58.7 ± 5.7 vs. 42.4 ± 14.7 × 109 /L, p = .0007, day 9), and did not reach 50 × 109 /L in several experiments. Platelet count increments correlated significantly with hemostatic efficacy for CS-PAS-PC transfusion only. DISCUSSION: CS-PAS-PCs might achieve similar hemostasis as RTS-PAS-PCs in thrombocytopenic patients with mild bleeding. Hemostatic efficacy could be improved by transfusing more CS-PAS-PCs.

Calcium Ion Chelation Preserves Platelet Function During Cold Storage.

OBJECTIVE: Platelet transfusion is a life-saving therapy to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. However, for >6 decades, safe and effective strategies for platelet storage have been an impediment to widespread use of platelet transfusion. Refrigerated platelets are cleared rapidly from circulation, precluding cold storage of platelets for transfusion. Consequently, platelets are stored at room temperature with an upper limit of 5 days due to risks of bacterial contamination and loss of platelet function. This practice severely limits platelet availability for transfusion. This study is to identify the mechanism of platelet clearance after cold storage and develop a method for platelet cold storage. Approach and Results: We found that rapid clearance of cold-stored platelets was largely due to integrin activation and apoptosis. Deficiency of integrin ß3 or caspase-3 prolonged cold-stored platelets in circulation. Pretreatment of platelets with EGTA, a cell impermeable calcium ion chelator, reversely inhibited cold storage-induced platelet activation and consequently prolonged circulation of cold-stored platelets. Moreover, transfusion of EGTA-treated, cold-stored platelets, but not room temperature-stored platelets, into the mice deficient in glycoprotein Ibα significantly shortened tail-bleeding times and diminished blood loss. CONCLUSIONS: Integrin activation and apoptosis is the underlying mechanism of rapid clearance of platelets after cold storage. Addition of a cell impermeable calcium ion chelator to platelet products is potentially a simple and effective method to enable cold storage of platelets for transfusion.

Antioxidant prevents clearance of hemostatically competent platelets after long-term cold storage.

BACKGROUND: Cold storage of platelets (PLTs) has the potential advantage of prolonging storage time while reducing posttransfusion infection given the decreased likelihood of bacterial outgrowth during storage and possibly beneficial effects in treating bleeding patients. However, cold storage reduces PLT survival through the induction of complex storage lesions, which are more accentuated when storage is prolonged. STUDY DESIGN AND METHODS: Whole blood-derived PLT-rich plasma concentrates from seven PLT pools (n = 5 donors per pool). PLT additive solution was added (67%/33% plasma) and the product was split into 50-mL bags. Split units were stored in the presence or absence of 1 mM of N-acetylcysteine (NAC) under agitation for up to 14 days at room temperature or in the cold and were analyzed for PLT activation, fibrinogen-dependent spreading, microparticle formation, mitochondrial respiratory activity, reactive oxygen species (ROS) generation, as well as in vivo survival and bleeding time correction in immunodeficient mice. RESULTS: Cold storage of PLTs for 7 days or longer induces significant PLT activation, cytoskeletal damage, impaired fibrinogen spreading, enhances mitochondrial metabolic decoupling and ROS generation, and increases macrophage-dependent phagocytosis and macrophage-independent clearance. Addition of NAC prevents PLT clearance and allows a correction of the prolonged bleeding time in thrombocytopenic, aspirin-treated, immunodeficient mice. CONCLUSIONS: Long-term cold storage induces mitochondrial uncoupling and increased proton leak and ROS generation. The resulting ROS is a crucial contributor to the increased macrophage-dependent and -independent clearance of functional PLTs and can be prevented by the antioxidant NAC in a magnesium-containing additive solution.

WoundClot® Hemostatic Gauze Reduces Bleeding Time after Arterial Venous Fistula Decannulation.

INTRODUCTION: Decannulation of the arteriovenous fistula (AVF) after each hemodialysis session requires a precise compression on the needle puncture site. The objective of our study was to evaluate the bleeding time (BT) needed to achieve hemostasis using WoundClot, an innovative hemostatic gauze, and to assess whether its long-term use can improve AVF preservation. METHODS: This is a prospective single center study. Initially, the time to hemostasis after AVF decannulation was compared between WoundClot and cotton gauze in 24 prevalent hemodialysis patients. Thereafter, the patients continued to use WoundClot for 12 months and were compared to a control group consisting of 25 patients using regular cotton gauze. Follow-up data included parameters of dialysis adequacy, AVF interventions, and thrombotic events. RESULTS: WoundClot use shortened significantly the time needed for hemostasis. Mean venous BT decreased by 3.99 (±4.6) min and mean arterial BT by 6.38 (±4.8) min when using WoundClot compared to cotton gauze (p < 0.001). At the end of the study, dialysis adequacy expressed by spKt/V was higher in the WoundClot group compared to control (1.73 vs. 1.53, respectively, p = 0.047). Although patients in WoundClot group had a higher baseline BT, arterial and venous pressures did not differ between the groups after a median follow up of 10.8 months. AVF thrombosis rate was similar between the groups. CONCLUSIONS: WoundClot hemostatic gauze significantly reduced the time required for hemostasis after AVF decannulation and may be associated with better AVF preservation. We suggest using WoundClot for arterial BT longer than 15 min and for venous BT longer than 12.5 min.

Absorbable Hemostatic Pack Effect After Primary Incisor Extraction: A Pilot Study and Introduction of a Novel Scale to Assess Post-Operative Bleeding.

OBJECTIVES: This pilot study compared hemostatic pack (HP) application with no intervention following extraction of maxillary primary incisors in healthy children for effect on bleeding time and influence of patient or tooth variables utilizing a novel scale for assessment of bleeding following extraction. STUDY DESIGN: A novel scale was created to assess bleeding after extraction. This scale was utilized in a randomized, split mouth study of healthy children ages 2-7 years old requiring extraction of at least 2 primary maxillary incisors under general anesthesia. One extraction site was randomly assigned to receive HP and the other had no hemostatic measures. Post-operative bleeding was rated at 2, 10, and 15 minutes post-extraction. Other variables recorded included age, sex, periapical radiolucency, presence of fistula, swelling, discoloration, intraoral stabilization device used, and vital signs at two time intervals. Pre-operative radiographs were reviewed for root resorption and periapical radiolucency. RESULTS AND CONCLUSIONS: Twenty-five patients provided 50 teeth. Hemostatic pack had a significant effect on reducing bleeding at each time point and that effect did not change over time. Age, sex, tooth pain, post-extraction heart rate, blood pressure, discoloration, amount of resorption, and presence of a periapical radiolucency had no significant effect on bleeding. The proposed bleeding scale had good intra-rater reliability and could be useful in future studies, once validated.

Short-Acting Anti-VWF (von Willebrand Factor) Aptamer Improves the Recovery, Survival, and Hemostatic Functions of Refrigerated Platelets.

OBJECTIVE: Refrigeration-induced binding of VWF (von Willebrand factor) to platelets contributes to the rapid clearance of refrigerated platelets. In this study, we investigate whether inhibiting VWF binding by a DNA-based aptamer ameliorates the clearance of refrigerated platelets without significantly impeding hemostatic functions. Approach and Results: Platelets were refrigerated with or without aptamer ARC1779 for 48 hours. VWF binding, the effective lifetime of ARC1779, platelet post-transfusion recovery and survival, and the hemostatic function were measured. ARC1779 treatment during refrigeration inhibited the platelet-VWF interaction. ARC1779-treated refrigerated murine platelets exhibited increased post-transfusion recovery and survival than untreated ones (recovery of ARC1779-treated platelets: 76.7±5.5%; untreated: 63.7±0.8%; P<0.01. Half-life: 31.4±2.36 hours versus 28.1±0.86 hours; P<0.05). A similar increase was observed for refrigerated human platelets (recovery: 49.4±4.4% versus 36.8±2.1%, P<0.01; half-life: 9.2±1.5 hours versus 8.7±0.9 hours, ns). The effective lifetime of ARC1779 in mice was 2 hours. Additionally, ARC1779 improved the long-term (2 hours after transfusion) hemostatic function of refrigerated platelets (tail bleeding time of mice transfused with ARC1779-treated refrigerated platelets: 160±65 seconds; untreated: 373±96 seconds; P<0.01). The addition of an ARC1779 antidote before transfusion improved the immediate (15 minutes after transfusion) hemostatic function (bleeding time of treated platelets: 149±21 seconds; untreated: 320±36 seconds; P<0.01). CONCLUSIONS: ARC1779 improves the post-transfusion recovery of refrigerated platelets and preserves the long-term hemostatic function of refrigerated platelets. These results suggest that a short-acting inhibitor of the platelet-VWF interaction may be a potential therapeutic option to improve refrigeration of platelets for transfusion treatment.

Effects of concomitant use of prasugrel with edoxaban on bleeding time, pharmacodynamics, and pharmacokinetics of edoxaban in healthy elderly Japanese male subjects: a clinical pharmacology study.

BACKGROUND: Dual therapy with a direct oral anticoagulant (DOAC) plus a P2Y12 receptor inhibitor is recommended in patients with nonvalvular atrial fibrillation who undergo percutaneous coronary intervention. Thus, we evaluated the effects of DOAC edoxaban plus P2Y12 receptor inhibitor prasugrel on bleeding time (BT), and pharmacodynamics (PD) and pharmacokinetics (PK) of edoxaban in healthy elderly Japanese male subjects. METHODS: A single-center, clinical pharmacology study with randomized, open-label, repeated dosing enrolled 24 participants in two groups of 12 receiving 30 mg edoxaban once daily for 3 days; then 30 mg edoxaban plus 2.5 mg prasugrel (Group 1) or 30 mg edoxaban plus 3.75 mg prasugrel (Group 2) once daily for 5 days. Primary endpoint was BT by the Ivy method. Secondary endpoints were the PD parameters of prothrombin time (PT), activated partial thromboplastin time (aPTT), prothrombin fragment F1 + 2 (F1 + 2), and P2Y12 reaction units (PRU), and PK profiles of edoxaban alone and in combination with prasugrel. RESULTS: Geometric least squares mean of BT ratios (vs. baseline) for 3-day edoxaban treatment were 1.097 (90% confidence interval (CI) 0.911-1.321) in Group 1 and 1.327 (90% CI 1.035-1.703) in Group 2; for 5-day edoxaban plus 2.5 mg and 3.75 mg prasugrel, they were 1.581 (90% CI 1.197-2.087) and 1.996 (90% CI 1.482-2.690), respectively. Contributions of prasugrel to the effects (edoxaban + prasugrel/edoxaban) were 1.442 (90% CI 1.096-1.897) in Group 1 and 1.504 (90% CI 1.172-1.930) in Group 2. Edoxaban prolonged PT and aPTT and decreased F1 + 2. Adding on prasugrel did not appreciably change PT, aPTT, or F1 + 2. Prasugrel reduced PRU, whereas edoxaban had no effect on PRU. We recorded 26 adverse events; 23 were treatment-emergent (positive fecal occult blood test). All participants with adverse events recovered during follow-up. CONCLUSIONS: Coadministration of 2.5 mg and 3.75 mg prasugrel with 30 mg edoxaban prolonged BT in healthy elderly Japanese male subjects. The effect was dependent on the dose of prasugrel. Prasugrel did not affect PD or PK profiles of edoxaban. Edoxaban had no effect on PD of prasugrel. TRIAL REGISTRATION: Japan Registry of Clinical Trials No. jRCTs071190006; registration date, 26-April-2019.

Association of the bleeding time test with aspects of traumatic brain injury in patients with alcohol use disorder.

BACKGROUND-AIM: Traumatic brain injury (TBI) and alcohol use disorder (AUD) can occur concomitantly and be associated with coagulopathy that influences TBI outcome. The use of bleeding time tests in TBI management is controversial. We hypothesized that in TBI patients with AUD, a prolonged bleeding time is associated with more severe injury and poor outcome. MATERIAL AND METHODS: Moderate and severe TBI patients with evidence of AUD were examined with bleeding time according to IVY bleeding time on admission during neurointensive care. Baseline clinical and radiological characteristics were recorded. A standardized IVY bleeding time test was determined by staff trained in the procedure. Bleeding time test results were divided into normal (≤ 600 s), prolonged (> 600 s), and markedly prolonged (≥ 900 s). Normal platelet count (PLT) was defined as > 150,000/µL. This cohort was compared with another group of TBI patients without evidence of AUD. RESULTS: Fifty-two patients with TBI and AUD were identified, and 121 TBI patients without any history of AUD were used as controls. PLT was low in 44.2% and bleeding time was prolonged in 69.2% of patients. Bleeding time values negatively correlated with PLT (p < 0.05). TBI patients with markedly prolonged values (≥ 900 s) had significantly increased hematoma size, and more frequently required intracranial pressure measurement and mechanical ventilation compared with those with bleeding times < 900 s (p < 0.05). Most patients (88%) with low platelet count had prolonged bleeding time. No difference in 6-month outcome between the bleeding time groups was observed (p > 0.05). Subjects with TBI and no evidence for AUD had lower bleeding time values and higher platelet count compared with those with TBI and history of AUD (p < 0.05). CONCLUSIONS: Although differences in the bleeding time values between TBI cohorts exist and prolonged values may be seen even in patients with normal platelet count, the bleeding test is a marker of primary hemostasis and platelet function with low specificity. However, it may provide an additional assessment in the interpretation of the overall status of TBI patients with AUD. Therefore, the bleeding time test should only be used in combination with the patient's bleeding history and careful assessment of other hematologic parameters.

Effect of Aspirin and ibuprofen either alone or in combination on gastric mucosa and bleeding time and on serum prostaglandin E2 and thromboxane A2 levels in the anaesthetized rats in vivo.

There is much evidence that a combination of ibuprofen (IBU) and Aspirin (ASA) can antagonize the irreversible inhibition of platelet function. This study was designed to investigate the degree of gastric damage, bleeding time (BT) and fluctuations in the serum levels of prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) after oral administration of ASA (200 mg/kg) and IBU (50 mg/kg) either alone or in combination in rats in vivo. The stomach was assessed for any damage either after 6 h, 18 h or 6 days and carboxymethylcellulose (1% CMC) served as a vehicle and control. ELISA was used to measure TXA2 and PGE2 in serum. Bleeding time was assessed using tail blood. The results show that ASA and IBU either alone or in combination can cause gastric ulceration in 25-100% of the rats at 6 and 18 h. In contrast, gastric ulceration was seen in 50% of rats with a combination of ASA given before IBU only after 6 days of oral administration. BT was unaffected either by ASA or IBU when administered alone except after 18 h for IBU. In contrast, BT was significantly reduced when IBU was administered before ASA after 18 h and 6 days (P < 0.001). Serum PGE2 levels decreased significantly after ASA administered either alone or in combination with IBU for 6 h, 18 h and 6 days (P < 0.05). Serum TXA2 levels were significantly reduced after 6 h, 18 h and 6 days following ASA and IBU administration except for IBU alone which caused a significant increase in serum TXA2 6 days after its administration (P < 0.01). It can be concluded that ASA and IBU administered either alone or in combination can cause gastric ulcers in the rat stomach after 6 h and 18 h, but less severe after 6 days. IBU either alone or in combination with ASA reduced BT only after 18 h and 6 days of administration. Together, the results show that gastric ulceration correlated well with the inhibition of serum PGE2 and TXA2 levels.

Trowaglerix Venom Polypeptides As a Novel Antithrombotic Agent by Targeting Immunoglobulin-Like Domains of Glycoprotein VI in Platelet.

OBJECTIVE: Currently prescribed antiplatelet drugs have 1 common side effect-an increased risk of hemorrhage and thrombocytopenia. On the contrary, bleeding defects associated with glycoprotein VI (GPVI) expression deficiency are usually slightly prolonged bleeding times. However, GPVI antagonists are lacking in clinic. APPROACH AND RESULTS: Using reverse-phase high-performance liquid chromatography and sequencing, we revealed the partial sequence of trowaglerix α subunit, a potent specific GPVI-targeting snaclec (snake venom C-type lectin protein). Hexapeptide (Troα6 [trowaglerix a chain hexapeptide, CKWMNV]) and decapeptide (Troα10) derived from trowaglerix specifically inhibited collagen-induced platelet aggregation through blocking platelet GPVI receptor. Computational peptide design helped to design a series of Troα6/Troα10 peptides. Protein docking studies on these decapeptides and GPVI suggest that Troα10 was bound at the lower surface of D1 domain and outer surface of D2 domain, which was at the different place of the collagen-binding site and the scFv (single-chain variable fragment) D2-binding site. The newly discovered site was confirmed by inhibitory effects of polyclonal antibodies on collagen-induced platelet aggregation. This indicates that D2 domain of GPVI is a novel and important binding epitope on GPVI-mediated platelet aggregation. Troα6/Troα10 displayed prominent inhibitory effect of thrombus formation in fluorescein sodium-induced platelet thrombus formation of mesenteric venules and ferric chloride-induced carotid artery injury thrombosis model without prolonging the in vivo bleeding time. CONCLUSIONS: We develop a novel antithrombotic peptides derived from trowaglerix that acts through GPVI antagonism with greater safety-no severe bleeding. The binding epitope of polypeptides on GPVI is novel and important. These hexa/decapeptides have therapeutic potential for developing ideal small-mass GPVI antagonists for arterial thrombogenic diseases.

Cubosomes as Oral Drug Delivery Systems: A Promising Approach for Enhancing the Release of Clopidogrel Bisulphate in the Intestine.

Clopidogrel bisulphate (CB) is a first line antiplatelet drug for treatment of myocardial infarction and stroke. Yet, its efficacy is limited by its poor solubility in intestinal pH, its main site of absorption. The main aim of this study is to enhance the intestinal release of CB by loading in cubosome nanoparticles. Glyceryl monooleate (GMO) based CB loaded cubosomes were prepared using a 33 factorial design to study the effect of polyvinyl alcohol (PVA), poloxamer 407 (PL407) concentrations and ratio of CB to the disperse phase on the average particle size, entrapment efficiency (%EE), in vitro release at 15 min (%Q15), and their morphology using transmission electron microscopy (TEM). The release of the optimized formula was compared in buffer transition media (pH 1.2 for 2 h then pH 6.8 for 6 h) to free drug to study the effect of the changing pH in the gastrointestinal tract (GIT) on CB release. The antihaemostatic properties of the optimized formula were compared to the commercial product Plavix® using bleeding time (BT) model in rabbits. The prepared cubosomes were in the nano range (115±6.47 to 248±4.63 nm) with high %EE (91.22±4.09% to 98.98±3.21%). The optimized formula showed significantly higher (p<0.05) CB release in intestinal pH and preserved the high% released (95.66±1.87%) in buffer transition release study compared to free drug (66.82±4.12%) as well as significantly (p<0.05) higher antihaemostatic properties with longer BT (628.47±6.12 s) compared to Plavix® (412.43±7.97 s). Thus, cubosomes proved to be a successful platform to enhance the intestinal release of CB and improve its absorption.

Novel Therapeutic Agent against Platelet Activation In Vitro and Arterial Thrombosis In Vivo by Morin Hydrate.

Morin hydrate, a bioactive flavonoid, has been proven to prevent inflammation and apoptosis of cells. Flavonoids can reduce the risk of cardiovascular diseases, in which platelet activation plays a major role. This study investigated the effect of morin hydrate on platelet activation in vitro and in vivo. Morin hydrate markedly inhibited platelet aggregation stimulated by collagen in human platelets but not that stimulated by other agonists. In collagen-activated platelets, morin hydrate inhibited adenosine triphosphate (ATP) release; intracellular Ca2+ mobilization; P-selectin expression; and phosphorylation of phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), and Akt. In mitogen-activated protein kinase (MAPK) activation, morin hydrate evidently diminished ERK2 or JNK1 activation, except for p38 MAPK. Additionally, morin hydrate markedly reduced the OH· signals in platelet suspensions but not in the cell-free system (Fenton reaction solution). Moreover, morin hydrate substantially increased the occlusion time of thrombotic platelet plug formation but had no effect on bleeding time in mice. In conclusion, morin hydrate crucially inhibits platelet activation through inhibition of the PLCγ2⁻PKC cascade and subsequent suppression of Akt and MAPK activation, thereby ultimately inhibiting platelet aggregation. Therefore, this paper suggests that morin hydrate constitutes a novel and potential natural therapeutic product for preventing or treating thromboembolic disorders.

Hemostatic effects of adrenaline and Ankaferd (blood stopper) during orthodontic attachment bonding

Background/aim: Moisture prevention during the bonding of orthodontic attachments on impacted teeth is crucial for accomplishment. It was aimed to compare the hemostatic effects of adrenaline and Ankaferd Blood Stopper (ABS) during the surgical exposure of the impacted maxillary canine. Materials and methods: The study consists of 20 patients, whose orthodontic treatments were outlined with the surgical exposure of maxillary impacted canine. Patients were divided into groups of 10; where each group was treated with one of the two medicines to control bleeding. Group A was treated with adrenaline, and group B was treated with Ankaferd Blood Stopper (ABS). The bleeding period was recorded as the time from the exposure of the crown until the inception of bonding. Results: It was observed that both the bleeding period and the cumulative duration were significantly shorter in group B (the ABS group) than in group A (the adrenaline group) (P < 0.05), but no significant deviation in bonding times was recorded. Conclusion: ABS is a good alternative hemostatic agent for the prevention of bleeding at the surgical exposure of impacted teeth without affecting the bonding.