Longer Bleeding Time Research Articles

Monoglyceride Lipase Deficiency Is Associated with Altered Thrombogenesis in Mice.

Monoglyceride lipase (MGL) hydrolyzes monoacylglycerols (MG) to glycerol and one fatty acid. Among the various MG species, MGL also degrades 2-arachidonoylglycerol, the most abundant endocannabinoid and potent activator of the cannabinoid receptors 1 and 2. We investigated the consequences of MGL deficiency on platelet function using systemic (Mgl-/-) and platelet-specific Mgl-deficient (platMgl-/-) mice. Despite comparable platelet morphology, loss of MGL was associated with decreased platelet aggregation and reduced response to collagen activation. This was reflected by reduced thrombus formation in vitro, accompanied by a longer bleeding time and a higher blood volume loss. Occlusion time after FeCl3-induced injury was markedly reduced in Mgl-/- mice, which is consistent with contraction of large aggregates and fewer small aggregates in vitro. The absence of any functional changes in platelets from platMgl-/- mice is in accordance with lipid degradation products or other molecules in the circulation, rather than platelet-specific effects, being responsible for the observed alterations in Mgl-/- mice. We conclude that genetic deletion of MGL is associated with altered thrombogenesis.

Management and Morbidity of Major Pelvic Hemorrhage in Complex Abdominopelvic Surgery

Introduction: Hemorrhage is a challenging complication of pelvic surgery. This study aimed to analyze the causes, management, and factors associated with morbidity in patients experiencing major pelvic hemorrhage during complex abdominopelvic surgery. Methods: Patients who had major intraoperative pelvic hemorrhage during complex abdominopelvic surgery at 11 tertiary referral centers between 1997 and 2017 were included. Patient characteristics, management strategies to control bleeding, short- and long-term postoperative outcomes were evaluated retrospectively. Results: There were 120 patients with a mean age of 56.6 ± 2.4 years and a mean BMI of 28.3 ± 1 kg/m². While 104 (95%) of the patients were operated for malignancy, 16 (5%) of the patients had surgery for a benign disease. The most common bleeding site was the presacral venous plexus 90 (75%). Major pelvic hemorrhage was managed simultaneously in 114 (95%) patients. Electrocauterization 27 (23%), pelvic packing 26 (22%), suturing 7 (6%), thumbtacks application 7 (6%), muscle welding 4 (4%), use of energy devices 2 (2%), and topical hemostatic agents 2 (2%) were the management tools. Combined techniques were used in 43 (36%) patients. Short-term morbidity and mortality rates were 48 (40%) and 2 (2%), respectively. High preoperative CRP levels (p = 0.04), history of preoperative radiotherapy (p = 0.04), longer bleeding time (p = 0.006), and increased blood transfusion (p = 0.005) were the factors associated with postoperative morbidity. Conclusion: Postoperative morbidity related to major pelvic hemorrhage can be reduced by optimizing the risk factors. Prehabilitation prior to surgery to moderate inflammatory status and prompt action with proper technique to control major pelvic hemorrhage can prevent excessive blood loss in complex abdominopelvic surgery.

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.

P2Y12 antibody inhibits platelet activity and protects against thrombogenesis

Given that platelet hyperactivity is known to give rise to thrombotic disorders, new and/or novel antiplatelet therapies are constantly being developed to add to, or to complement the current arsenal of agents. To this end, adenosine diphosphate (ADP) is an important platelet activator that acts by binding to the G-protein coupled P2Y1 and P2Y12 receptors. Although the contribution of the P2Y12 receptor to the genesis of thrombosis is well established, the parenteral arsenal of drugs targeting this receptor in clinical use is limited to cangrelor. In this study, we investigated the potential antiplatelet activity of an antibody targeting the ligand-binding domain of the P2Y12 receptor (abbreviated P2Y12Ab). Our in vitro studies revealed that the P2Y12Ab could effectively inhibit aggregation induced by ADP, as well as that triggered by the thromboxane receptor agonist U46619. Additionally, using FACS analysis, we observed reduced P-selectin, phosphatidylserine exposure and integrin activation in the presence of P2Y12Ab. As for its in vivo effects, the P2Y12Ab also demonstrated protection against thrombus formation; albeit this was accompanied with a bleeding diathesis (longer bleeding time). Notably, this inhibitory profile is consistent with that observed with oral anti-P2Y12 agents. Collectively, our findings demonstrate that the P2Y12Ab functionally blocks platelet activity in vitro and in vivo, and support the notion that it can be purposed as a parenteral antiplatelet agent, to be used in conjunction with and/or as a complement to current antiplatelet therapies.

Abstract 351: Plasma Phospholipid Transfer Protein Promotes Platelet Aggregation

Hypothesis: Phospholipid transfer protein (PLTP) has a direct effect on platelet aggregation, since PLTP knockout mice have longer bleeding time. Methods and Results: Platelets from humans or mice were prepared as were mouse platelet-rich plasma and human recombinant PLTP (rPLTP). In mice, we assessed ADP- and collagen-induced platelet aggregation, phosphatidylserine (PS) externalization, and photothrombosis-induced cerebral infarction. We found that human platelets produce PLTP. Platelet aggregation increased upon PLTP overexpression whereas it decreased with PLTP deficiency in a gene dose-dependent manner. Human rPLTP increased mouse or human platelet aggregation in a dose-dependent manner. PS externalization provides a water/lipid surface for the interaction of coagulation factors, which accelerates thrombosis. Compared with wild type controls, platelets from PLTP transgenic mice had significantly greater amounts of PS on the exterior surface of the plasma membrane, whereas platelets from PLTP-deficient mice had significantly less on the surface, thus influencing fibrinogen binding. Moreover, rPLTP together with ADP significantly increased PS exposure on the plasma membrane of PLTP-deficient platelets, thereby increasing fibrinogen binding. Importantly, PLTP overexpression significantly accelerated the incidence of photothrombosis-induced infarction, whereas PLTP deficiency reduced the incidence. Conclusions: PLTP promotes PS externalization at the plasma membrane of platelets and accelerates ADP- or collagen-induced platelet aggregation. Thus, PLTP is involved in hypercoagulation. Therefore, PLTP inhibition could be a novel approach for countering thrombosis.

Differential anti-thrombotic benefit and bleeding risk profiles of antagonists of protease-activated receptor 1 and 4 in Cynomolgus Macaques

Platelet activation plays a crucial role in hemostasis and thrombosis. Thrombin, the most potent stimulus of platelet activation, mediates platelet activation via the protease activated receptors (PARs). The platelet PAR repertoire in mediating thrombin's action differs across species. Only nonhuman primate (NHP) platelet activation is known to be similar to humans, mediated by PAR1 and PAR4, hence limiting translational in vivo studies of PAR's role in thrombosis and hemostasis to NHPs. Earlier studies have demonstrated a range of distinct in vitro activities of PAR1 and 4 in platelet activation yet the implications of these events in vivo is unclear. The objective of this study is to investigate and compare the roles of PAR1 and PAR4 in hemostasis and thrombosis in a relevant animal species. NHP models for pharmacokinetic, ex vivo platelet aggregation responses, FeCI3 injury-mediated arterial thrombosis and template bleeding were developed in Cynomolgus Macaques. Potent and selective small molecule antagonists of PAR1 and PAR4 were characterized in an array of in vitro assays, and subsequently examined head-to-head in the NHP models. Treatment of NHPs with antagonists of PAR1 or PAR4 both resulted in strong inhibition of ex vivo platelet aggregation. At doses that led to similar inhibition of platelet aggregation, animals treated with the PAR4 antagonist showed similar levels of anti-thrombotic efficacy, but longer bleeding times, compared to animals treated with the PAR1 antagonist. These findings suggest that PAR1 antagonism will likely produce a larger therapeutic index (ie. a larger anti-thrombotic efficacy over bleeding risk margin) than PAR4 antagonism.

Prasugrel Versus Clopidogrel: A Comparative Examination of Local Bleeding After Dental Extraction in Patients Receiving Dual Antiplatelet Therapy

To study the effects of various parameters on local hemostasis after dental extraction in patients receiving different combinations of medications who had previously confirmed effective dual inhibition of platelet aggregation. A total of 129 patients were enrolled. They underwent acute or planned percutaneous coronary intervention and their stomatological examination disclosed teeth that could have acted as foci and thus had to be removed. All patients took acetylsalicylic acid 100 mg and clopidogrel or prasugrel. Lidocaine with or without epinephrine was used for local anesthesia, and a gauze swab or suture was applied to help hemostasis. Bleeding time was significantly longer by an average of 10 minutes (+21%) in patients taking prasugrel (P < .05) compared with those taking clopidogrel. Use of a suture resulted in a significantly shorter bleeding time after anesthesia with or without epinephrine (P < .05). A considerably longer bleeding time was observed when anesthesia with no epinephrine was combined with gauze. In smokers, the bleeding time was shorter by 15% on average. This study is the first to analyze differences in bleeding times between clopidogrel and prasugrel treatments during dental extraction. In general, prasugrel is associated with a considerably longer bleeding time; nevertheless, dental extraction can be performed safely with either combination.

Quantifying avoidance-related behaviour and bleeding times of sheep of different ages, sex and breeds slaughtered at a municipal and a commercial abattoirs

The effect of abattoir type, age, sheep breed and sex on avoidance-related behaviour and bleeding times at slaughter were determined. Castrates and ewes of different age groups and breeds were used. From the commercial abattoir, 90 castrates and 110 ewes were used while 66 castrates and 19 ewes were obtained from the municipal abattoir. Avoidance-related behaviour and bleeding times differed with abattoir type and age classes. Animals slaughtered at the commercial abattoir were calmer and had shorter bleeding times (61 ± 1.16 s) than those slaughtered at the municipal abattoir which had a mean bleeding time of 74.2 ± 2.11 s. Older animals were calmer and had longer bleeding times (67.5 ± 1.82 s) than the younger animals which had a mean bleeding time of 61.6 ± 2.64 s. Avoidance-related behaviour also differed with breed and the sex of the animals with Merino and Dorper + Merino crosses being the calmer than the pure Dorper breed. About 95% of ewes were calmer than the castrates. It can be concluded that abattoir type, age, breed and sex have an effect on animal behaviour at slaughter. Only abattoir type and sex had an effect on bleeding times.

Acute dysphagia after myocardial infarction: an unusual complication of anticoagulation therapy

Antiplatelet medications such as aspirin, clopidogrel or prasugrel are commonly used in patients with myocardial infarction (MI) and stent implantation. Additionally, thrombolytic therapy is recommended for patients in whom a coronary intervention cannot be performed in a timely manner. Both classes of drugs put patients at risk for major bleeding complications. However, also unusual bleeding complications can lead to serious complications. A 55-year-old male contacted the emergency medical service with acute onset of chest pain beginning 1.5 h before. The ECG showed typical ST-segment elevations (Fig. 1). An inferior MI was diagnosed, and a pre-hospital thrombolytic therapy with 10,000 units of tenecteplase was administered. On admission to our hospital, the chest pain was still present but reduced. The ECG showed persistent ST-segment elevations and a rescue percutaneous coronary intervention was performed. An additional 6,000 IE of heparin were given for the intervention. An acute occlusion of the right coronary artery was successfully reopened and a drug-eluting stent (tacrolimus-eluting-stent, TAXUS®, Boston Scientific) was placed (Fig. 2). In the clinical course, cardiac ultrasound showed a moderate impairment of left ventricular function (LVEF 40%). The patient was treated with aspirin 100 mg/d and prasugrel 10 mg/d. Two days later, the patient developed a progressively sore throat and reported new-onset dysphagia. Within the next few hours, a progressive swelling of the left cervical region occurred. Emergency computed tomography of the neck showed a massive left sided tumour, consistent with a large bleeding into a thyroidal cyst. The diameters of the haematoma were 13 × 7 × 6 cm, with displacement and compression of trachea to 6 mm (Fig. 3). To secure the airways, the patient was endotracheally intubated without problems. An immediate left hemithyreoidectomy with decompression of the haematoma was performed. The patient recovered quickly after the operation. Histological findings showed no evidence of malignancy. Spontaneous haemorrhage into a thyroid cyst is a rare condition. When causing airway obstruction, this life-threatening situation requires immediate airway protection before complete obstruction occurs. In the literature, some cases of retropharyngeal or thyreoideal haematoma have been reported as complications of medical manoeuvres such as intubation or thyroid puncture, or as a complication of chronic anticoagulation therapy with warfarin or marcumar. To the best of our knowledge, this is the first description of a spontaneous haemorrhage into a thyroid cyst under aspirin and prasugrel. Prasugrel is known to lead to more pronounced platelet inhibition than clopidogrel, resulting in a four to five times longer bleeding time and a faster onset of action. Clearly, the faster and more effective platelet inhibition obtained by prasugrel may lead to an increased risk of major and even atypical bleeding events after previous fibrinolytic therapy.

Safety, tolerability, pharmacokinetics and pharmacodynamics of high single-ascending doses of ticagrelor in healthy volunteers

Previous studies have indicated that ticagrelor is well tolerated and exhibits linear pharmacokinetics up to doses of 600 mg/day. The safety, tolerability, pharmacokinetics and pharmacodynamics (bleeding times and pulmonary function tests) of high single-ascending doses of ticagrelor were assessed to determine the maximum tolerated dose of ticagrelor. This was a randomized, double-blind, placebo-controlled study. Eight healthy volunteers were planned for enrollment in each of 3 dose groups, ticagrelor 900 mg, 1,260 mg, and 1,620 mg (6 : 2 ratio ticagrelor : placebo). The study stopping criteria were met when 3 of the 6 volunteers receiving ticagrelor 1,260 mg experienced moderate gastrointestinal adverse events (AE); none were observed with placebo. One volunteer receiving ticagrelor 1,260 mg had a serious AE - sinus arrest, high-grade atrioventricular block, and ventricular escape rhythm with syncope - and another volunteer had brief, mild dyspnea. Ticagrelor 900 mg was well tolerated. Total exposure to ticagrelor increased dose proportionally. Peak plasma concentration (Cmax) for ticagrelor did not increase much, most likely due to delayed absorption. There were no relevant changes in respiratory parameters. Bleeding times were prolonged in those receiving ticagrelor with respect to placebo, with longer bleeding times in volunteers receiving ticagrelor 1,260 mg than in volunteers receiving 900 mg; no bleeding events were reported. These results indicate that the maximum tolerated single dose of ticagrelor is 900 mg in healthy volunteers.

Bleeding tendency in dual antiplatelet therapy with aspirin/clopidogrel: rescue of the template bleeding time in a single-center prospective study

BackgroundPatients with heightened platelet reactivity in response to antiplatelet agents are at an increased risk of recurrent ischemic events. However, there is a lack of diagnostic criteria for increased response to combined aspirin/clopidogrel therapy. The challenge is to identify patients at risk of bleeding. This study sought to characterize bleeding tendency in patients treated with aspirin and clopidogrel.Patients/methodsIn a single-center prospective study, 100 patients under long-term aspirin/clopidogrel treatment, the effect of therapy was assayed by template bleeding time (BT) and the inhibition of platelet aggregation (IPA) by light transmission aggregometry (LTA). Arachidonic acid (0.625 mmol/L) and adenosine diphosphate (ADP; 2, 4, and 8 μmol/L) were used as platelet agonists.ResultsBleeding episodes (28 nuisance, 2 hematuria [1 severe], 1 severe proctorrhagia, 1 severe epistaxis) were significantly more frequent in patients with longer BT. Template BT ≥ 24 min was associated with bleeding episodes (28 of 32). Risk of bleeding increased 17.4% for each 1 min increase in BT. Correlation was found between BT and IPAmax in response to ADP 2 μmol/L but not to ADP 4 or 8 μmol/L.ConclusionIn patients treated with dual aspirin/clopidogrel therapy, nuisance and internal bleeding were significantly associated with template BT and with IPAmax in response to ADP 2 μmol/L but not in response to ADP 4 μmol/L or 8 μmol/L.

Template bleeding times of 240 neonates born at 24 to 41 weeks gestation

The template bleeding time is a test used to assess the hemostatic effectiveness of platelet/endothelial interactions. A modified template bleeding time, described over 15 years ago by Andrew et al., has been used to test this aspect of hemostasis in term and preterm infants, but questions remain regarding the effect of gestational age and postnatal age on results. The effect of the platelet count (platelets per microliter blood) and the circulating platelet mass (nl platelets per microliter blood) on the bleeding time of neonatal intensive care unit (NICU) patients also require better definition. We measured template bleeding times on 240 neonates at Ospedale A Perrino, in Brindisi, Italy; studying groups of n=60 at the following gestational ages; <28 weeks, 29 to 32 weeks, 33 to 37 weeks and > or =38 weeks. In each group of 60 neonates, 20 were studied on the first day after birth, 20 were studied on day 10 and 20 were studied on day 30. A multivariate analysis was performed to examine various associations with bleeding time. Bleeding times, on the first day of life, were shorter as gestational age increased. Those born <33 weeks gestation had bleeding times about twice that of those > or =38 weeks (P<0.001). Bleeding times tended to shorten between days 1 and 10. Little or no further shortening occurred between days 10 and 30, and by day 30 they were not statistically different between the various gestational age groups. No independent effect on bleeding time could be ascribed to gender, platelet count or circulating platelet mass, but independent effects were found for hematocrit (P<0.02) and gestational age (P<0.001). On the first day of life, preterm neonates have a longer bleeding time than do term neonates. By day of life 10, the bleeding times at all gestational ages are shorter and are indistinguishable on the basis of gestational age at birth. Additionally, since platelet counts as low as 110 000 per microliter did not prolong the bleeding time, we see no benefit of administering a platelet transfusion to a stable, nonbleeding, NICU patient with mild thrombocytopenia (platelet count 100 000 to 150 000 per microliter).

Platelet Dysfunction in Patients with Severe Traumatic Brain Injury

Coagulopathy is a common phenomenon in traumatic brain injury (TBI) and a major contributor to a poor outcome. Thrombocytopenia is a strong negative prognostic factor in TBI, but bleeding tendency can be present even with a normal platelet count. We investigated platelet function in patients with TBI by means of modified thromboelastography (i.e., platelet mapping [TEG-PM]). Four groups were studied: (1) patients with severe isolated TBI (n = 20), (2) patients with general trauma without TBI (the ICU group, n = 10), (3) patients with chronic alcohol abuse (n = 7; as alcohol abuse is common in patients with TBI), and (4) healthy volunteers (n = 10). We measured platelet counts in venous blood (Plt), Ivy bleeding time, standard TEG parameters, and platelet responses to arachidonic acid (AA) and adenosindiphosphate (ADP), using TEG-PM. TBI patients had a lower Plt (180 +/- 68 x 10(9) ; mean +/- SD) and a longer bleeding time (674 +/- 230 sec) than healthy controls, (256 +/- 43 x 10(9), p < 0.01) and (320 +/- 95 sec, p < 0.005), respectively. TBI patients had dramatically lower platelet responses to AA (0-86%, mean 22%) compared to healthy controls (57-89%, mean 73%), the ICU group (4-75%, mean 49%), and the alcohol abusers (17-88%, mean 64%; p < 0.001). Responses to ADP did not differ significantly between the groups. Patients with low responsiveness to AA at admittance to the hospital were likely to develop bleeding complications later. Patients with TBI develop platelet dysfunction, which most likely contributes to bleeding complications. The observed platelet dysfunction appears to involve the cyclooxygenase pathway. TEG-PM analysis can be used to identify patients with a high risk of bleeding complications.

Bleeding after medication-induced termination of pregnancy with two dosing schedules of mifepristone and misoprostol

Objective This study examines bleeding pattern following medication-induced termination of pregnancy, comparing two different dosing schedules of mifepristone and misoprostol. Study Design Diary information was analyzed from a randomized, multicenter trial in which women used vaginal misoprostol 800 μg either 6–8 or 24 h following 200 mg of oral mifepristone. Participants and Methods One thousand eighty women with pregnancies up to 63 days' gestation were recruited for the study; 540 were randomized to the 6- to 8-h dosing schedule, and 540 were randomized to the 24-h dosing schedule. Subjects recorded daily bleeding in a diary over 5 weeks. Results Total duration of bleeding ranged from 1 to 54 days, with a median of 7 days. Duration of spotting ranged from 1 to 80 days, with a median of 5 and 6 days (NS) in each of the two groups. Neither duration of bleeding nor duration of spotting were related to interval between mifepristone and misoprostol. Bleeding and spotting durations were not correlated with maternal age or smoking. Increased gestational age was correlated with longer bleeding and spotting times. Nulliparity was associated with longer bleeding time. Conclusion Varying the interval between mifepristone and misoprostol in medication abortion does not affect duration or quantity of bleeding.

Thromboxane and prostacyclin biosynthesis in patients with acute spontaneous intracerebral hemorrhage

Objective: Elevated levels of 11-dehydrothromboxane B 2 (11-dehydro-TXB 2) excreted in urine have been observed in acute ischemic stroke. This marker of platelet activation has not been investigated in patients with acute spontaneous intracerebral hemorrhage (ICH). Methods: We examined 43 patients with spontaneous ICH and 23 controls. Urinary excretion rates of 11-dehydro-TXB 2, 2,3-dinor-thromboxane B 2 (2,3 dinor-TXB 2) and 2,3-dinor-6-ketoprostaglandin F 1α (2,3-dinor-PGF 1α) during the first week and at 3 months after ICH were compared between patients who had or had not used aspirin and controls. Results: On admission, ICH patients without aspirin use had significantly higher urinary levels of 11-dehydro-TXB 2 ( p<0.001), 2,3-dinor-TXB 2 ( p<0.001) and 2,3-dinor-PGF 1α ( p=0.019) than controls. Aspirin users had significantly lower urinary levels of these metabolites than nonusers. The metabolite levels of aspirin users on admission did not significantly differ from those of controls. The differences between aspirin users and nonusers leveled off during the following 3–5 days, however, as the blocking effect of aspirin on the production of TXA 2 and PGI 2 ceased. Three months after ICH, the metabolite excretion levels in all the patients were similar to those in nonusers of aspirin on admission. On admission, aspirin users had longer bleeding times ( p=0.032) than nonusers, but aspirin use did not associate with impaired recovery or hematoma enlargement. Conclusions: Urinary excretion levels of 11-dehydro-TXB 2, 2,3-dinor-TXB 2 and 2,3-dinor-PGF 1α were higher in patients with acute ICH than in controls. The levels in aspirin users were equally low as in controls but rose to the levels of the other patients within a few days. The metabolite levels remained high 3 months after ICH in all patients. Prior use of aspirin did not seem to cause hematoma enlargement.

The relationship between hematocrit and bleeding time in very low birth weight infants during the first week of life.

The bleeding time is a measurement of platelet and capillary interaction following a small standardized cutaneous incision. In adults, anemia causes a prolongation of the bleeding time, and we hypothesized that the same would be true in very low birth weight (VLBW) infants during their first week of life. Template bleeding times, using the Surgicutt Newborn device, were performed on 20 VLBW weight infants <or=7 days old, before, and again following a clinically ordered erythrocyte transfusion. Neonates who had pretransfusion hematocrits <or=0.28 l/l had longer bleeding times, which fell 164+/-25 seconds (mean+/-SD; p<0.0001) following transfusion. Patients with pretransfusion hematocrits >0.28 l/l had no significant reduction in bleeding time following transfusion. In VLBW infants, during their first week of life (the time when their risk of intraventricular hemorrhage is greatest), a low hematocrit is associated with a significant prolongation in the bleeding time.

Bleeding tendency in Wolfram syndrome: a newly identified feature with phenotype genotype correlation.

Wolfram syndrome (WS) is a recessively inherited disorder associated with recognised clinical features. Bleeding tendency was noticed in some of our patients, although this has not been reported before. We therefore studied this problem in all our WS patients and tried to postulate a possible pathogenesis. At the same time, a genetic linkage study provided evidence of locus heterogeneity of this syndrome and showed that the majority of our patients belong to the second WS locus identified in that study. Our study group consisted of 13 WS patients, belonging to WSF2 locus (group I). Controls consisted of 4 healthy siblings of WS patients (group II) and 7 diabetics who do not have WS (group III). Relevant clinical data were obtained, and a coagulation screen was carried out for all groups. All individuals in the three study groups have normal platelet count, thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (aPTT), clot retraction, Factor VIII activity (FVIIIc) and von Willebrand factor antigen (vWAg). Eleven of the WS patients have prolonged template bleeding time (BT) compared with both control groups. Patients with WS have a longer BT (mean 9.6 min, 95% CL 8.61-10.53 min) than the siblings group (mean 6.75 min, 95% CL 5.52-7.98 min) and the diabetic group (mean 5.49 min, 95% CL 4.56-6.42 min). The differences between the study group and controls are statistically significant, p = 0.02 and 0.0002, respectively. In the three groups, platelet aggregation studies were normal using adenosine diphosphate (ADP), ristocetin and epinephrine. Aggregation with collagen was either absent or impaired, with failure of secondary wave being noticed in 11 of the WS patients (85%) and normal in the control groups. The pathogenesis of this problem is not known, but could be due to an inhibitory effect of vWAgII, deficiency of thrombospondin or a defect in the platelet membrane GPIa/IIa. Bleeding diathesis is a new additional feature to the clinical spectrum of WS, which is probably a feature of the disorder WFS2 and not WFS1, as bleeding has never been reported in the latter. This provides further evidence for the phenotypic and genotypic heterogeneity of this complex disorder and may provide clues to the search for the second gene responsible for this phenotype.

Prostacyclin synthase gene transfer inhibits neointimal formation in rat balloon-injured arteries without bleeding complications.

This study was designed to compare the effects of prostacyclin synthase (PCS) gene transfer with those of a systemic infusion of beraprost sodium (BPS), a prostacyclin analogue, on vascular smooth muscle cell (VSMC) proliferation and neointimal formation after arterial injury. PCS gene (3 or 30 micrograms) was transfected into rat balloon-injured carotid arteries by a non-viral lipotransfection method. BPS (100 or 300 micrograms/kg/day) was subcutaneously infused with osmotic pumps after the injury. LacZ gene (30 micrograms) was used as a control. VSMC proliferation was estimated by the bromodeoxyuridine (BrdU) index (BrdU-positive nuclei/total nuclei) at day 7. Neointimal formation was evaluated at day 14. Each treatment group had six rats. PCS gene transfer prevented the increase in intimal/medial area ratio (3 micrograms: 46.6%, 30 micrograms: 61.1% reduction; P < 0.05, P < 0.01, respectively), as did BPS 300 micrograms/kg/day (49.8% reduction; P < 0.05). BPS 100 micrograms/kg/day, however, had no effects on the ratio. PCS gene transfer and BPS 300 micrograms/kg/day significantly suppressed the BrdU index. BPS 300 micrograms/kg/day group had more frequent hematoma and longer bleeding time. There were no significant differences in blood pressure, heart rate, or urinary volume among all groups. Both PCS gene transfer and BPS 300 micrograms/kg/day reduced neointimal formation after arterial injury by inhibiting VSMC proliferation. PCS gene transfer may be a safer therapeutic modality against neointimal formation than a systemic infusion of BPS because the former method resulted in fewer bleeding complications.

Prolonged bleeding time: a new clinical manifestation of hepatocellular carcinoma?

The association between prolonged bleeding time and hepatocellular carcinoma (HCC) has not been well studied. We investigated whether bleeding time is prolonged in cirrhotic patients with HCC and studied the role of clinical characteristics, tumour size, and laboratory data in predicting bleeding time prolongation. After excluding patients that presented with blood dyscrasia and uraemia, 58 cirrhotic patients with HCC, 106 cirrhotic patients without HCC, and 44 age-and sex-matched healthy subjects were included in the study. Bleeding time, imaging studies, clinical characteristics and biochemical data were obtained for every patient. Cirrhotic patients with and without HCC had longer bleeding times (554 +/- 32 s, respectively) compared with healthy controls (357 +/- 13 s, P < 0.05). Hepatocellular carcinoma patients with a large tumour burden (> 5 cm in diameter) had a significantly longer bleeding time than those patients without (663 +/- 105 vs 376 +/- 23 s, respectively, P < 0.05). After excluding patients with a platelet count < or = 80 000/mm3, cirrhotic patients classified as Child-Pugh's grading A and with a large tumour burden had longer bleeding times(580 +/- 87 s) than patients with a small tumour burden (< or = 5cm in diameter) and cirrhotic patients without HCC (371 +/- 22 and 416 +/- 29 s, respectively, P < 0.05). In cirrhotic patients with HCC, higher serum bilirubin levels, a Child-Pugh's grading C, and a tumour size > 5 cm in diameter were found to be significant predictors for prolonged bleeding time on univariate analysis. On multivariate analysis, both tumour size > 5 cm in diameter and a Child-Pugh's grading C (odd's ratio, 95% confidence interval and P value were measured as 38.5, 2.8-534.7, < 0.001, and 10.5, 0.9-117.6, 0.02, respectively) were the significant independent predictors. A significant correlation existed between tumour diameter and bleeding time (r = 0.44, P < 0.01). In conclusion, these results suggest that prolonged bleeding time may be categorized as a new clinical manifestation in patients with HCC. In addition to cirrhosis, HCC itself may also participate in the pathogenesis of bleeding time prolongation.

Hemostatic and fibrinolytic response to desmopressin in uremic patients.

Desmopressin (DDAVP) 0.3 micrograms/kg was infused in 20 uremic patients with prolong bleeding time. Prior to infusion, the uremic patients had a reduced level of tissue plasminogen activator (t-PA), normal levels of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor activity (vWF:RCo) and elevated level of factor VIII coagulant activity (FVIII:C). Patients with lower hematocrit or t-PA levels tended to have a longer bleeding time. One hour after DDAVP infusion, the bleeding time was shortened significantly. This improvement was significant in all patient groups irrespective of the high or low initial levels of factor VIII complex components. Plasma levels of FVIII:C, vWF:Ag, vWF:RCo and t-PA all increased significantly. The magnitude of increase in these factors, however, was not significantly correlated with the extent of bleeding time shortening. The multiple regression model for predicting the extent of bleeding time shortening suggested only two variables, viz initial bleeding time and posttreatment FVIII:C activity to be of significance. The present results indicate that the hemostatic response to DDAVP is uniform in uremic patients, regardless of whether the initial activities of factor VIII complex components are high or low. Posttreatment FVIII:C activity appears to play a significant role in the hemostatic action of DDAVP. Furthermore, a depressed fibrinolytic activity was generally observed to concur with the hemostatic defect in uremic patients.