Mean Clotting Time Research Articles

Metabolic effects of contraceptive steroids. Pt. 2. Blood coagulation.

24 healthy women aged 18 to 45 years of age from the Family Planning Clinic were divided into 2 matched groups (matched as to age, parity, weight, nutritional status, and interval since the last childbirth). One group was given a combination type tablet (Noracyclin 1 mg) and the other group, another combination tablet (Voldys). Coagulation status was assessed 1) during the initial control cycle before the drugs were started, and 2) between the 22nd to 26th day of 1st, 3rd and 5th cycles during treatment. Mean clotting time declined in both groups, with the changes being statistically significant in the Voldys group when initial levels are compared to those of the 3rd cycle and 5th cycle (p 0.05). Mean prothrombin time decreased significantly (p 0.05) with Voldys when control and 3rd cycle values are compared, but the values tended to reach pretreatment levels by the 5th cycle. There is a tendency for mean recalcification time to decline in both types of drugs. No significant differences were observed in the mean fibrinogen level and mean platelet counts in both groups. Generally, with Noracyclin, significant increase was seen only in platelet adhesiveness. With Voldys, clotting time and recalcification time was significantly decreased, and the prothrombin time though exhibiting a significant decrease in 3rd treatment cycle, reverted to pretreatment levels in the 5th cycle.

Evaluation of platelet function screening tests to detect platelet procoagulant deficiency in dogs with Scott syndrome

Clinical diagnosis of platelet dysfunction is complex and technically challenging. The wide repertoire of platelet responses requires a test panel to assess different parameters of platelet reactivity. While "global" hemostasis analyzers and whole blood assays have potential for testing platelet function, their ability to evaluate platelet procoagulant activity is ill-defined. The aim of this study was to determine whether platelet procoagulant deficiency, the pathophysiologic defect of Scott syndrome, could be detected in point-of-care and whole blood assays. Study subjects were 4 Scott syndrome-affected German Shepherds and 8 control dogs. We evaluated 2 point-of-care instruments: the platelet function analyzer (PFA-100) and thromboelastograph (TEG). TEG analysis was performed on recalcified citrated whole blood with and without tissue-factor activation. A whole blood flow cytometric assay was configured to detect thrombin-induced platelet P-selectin expression and platelet-derived microparticle release. Cytometric samples were analyzed after 1 hour and 1 day of storage. We found no significant differences between Scott and control dogs in PFA-100 COL/ADP closure times or in any TEG parameter in tissue-factor-activated samples. In nonactivated samples, mean clotting time (K) and time to maximal rate of thrombus generation were significantly prolonged in Scott dogs; however, values overlapped with those of control dogs. Cytometric analysis of samples from Scott dogs showed significantly diminished platelet-derived microparticle release. Samples from all dogs reanalyzed after 1 day of storage had nonspecific increases in basal P-selectin expression and vesiculation. A whole blood cytometric assay to detect stimulated platelet microparticle release can be used to screen for Scott syndrome. However, platelet activation artifacts preclude overnight storage for next-day analysis.

Exercise-Induced Hemostatic Alterations Are Detectable by Rotation Thrombelastography (ROTEM): A Marathon Study

Rotation thrombelastography (ROTEM) provides a whole blood assay that allows the assessment of plasmic- and platelet-related hemostasis in a single-step procedure. In our current study, we focused on the capability of the method to detect hemostatic alterations induced by physical exercise, enrolling 33 healthy participants of the Dusseldorf Marathon 2006. Venous blood drawn immediately before and after finishing the marathon was analyzed by a rotational thrombelastograph (Pentapharm, Munich, Germany). On initiation of blood coagulation by recalcification, standard ROTEM parameters were determined. Comparison of the results obtained before and after the physical exercise was performed using the Student t test for paired samples. As a result, the mean clotting time (CT) determined from blood samples obtained immediately after the marathon was significantly shorter (662.9 + or - 67.8 seconds vs 505.6 + or - 97.3 seconds, P = .002) and the mean maximal clot firmness was significantly broader (48.4 +/- 6.6 mm vs 51.5 +/- 4.5 mm, P = .0004) when compared to results obtained before the physical exercise. Differences between mean clot formation times (CFTs; 280.6 + 96 seconds vs 270.4 + or - 73.8 seconds) and mean alpha angles (45.9 degrees + or - 8 degrees vs 47.8 degrees + or - 5.8 degrees ) before and after the marathon were not statistically significant. Remarkably, some participants showed opposed results, particularly prolongation of CT and narrowing of maximum clot firmness (MCF). Our study demonstrates that ROTEM is sensitive to exercise-induced hemostatic alterations. The method appears to be capable of detecting even distinct changes in hemostasis in a single-step procedure. Further analyses are needed to clarify which hemostasis parameters influence ROTEM results and which ROTEM results are independent predictors of exercise-induced alterations of plasmic and platelet function. This might help to explain interindividual differences in exercise-induced alterations of hemostasis.

Exodontia and Antiplatelet Therapy

The fear of excessive bleeding often prompts the physician to stop long-term, low-dose antiplatelet therapy before any surgical procedure. This may put the patient at risk of an adverse thromboembolic event. We undertook an assessment of the incidence of prolonged postoperative bleeding after dental extractions among patients on uninterrupted antiplatelet therapy, and evaluated the need to stop such medications before dental extractions. Eighty-two patients requiring dental extractions were included in this study, of whom 57 were on antiplatelet therapy (aspirin). Patients were divided into 3 groups. Group 1 consisted of patients in whom antiplatelet therapy was interrupted (n = 25), group 2 consisted of those continuing their medication (n = 32), and group 3 comprised healthy patients not on antiplatelet therapy (n = 25). Preoperative bleeding time and clotting time were determined in all patients. The surgical procedure involved single or multiple teeth extractions under local anesthesia with a vasoconstrictor. All patient groups were similar regarding age, gender distribution, dosage of antiplatelet drug, and medical condition for which the drug was prescribed. Events of single or multiple teeth extractions were also comparable among the 3 groups. Pressure packing was performed in all cases as in routine dental extractions. One-way analysis of variance was performed to determine the significance of prolonged bleeding among groups. The mean bleeding times in groups 1, 2, and 3 were 3 minutes, 2 minutes and 45 seconds, and 1 minute and 49 seconds, respectively. The mean clotting times in groups 1, 2, and 3 were 5 minutes and 4 seconds, 4 minutes and 52 seconds, and 3 minutes and 42 seconds, respectively. No patient in any group had any episode of prolonged or significant bleeding from the extraction sites. Local hemostasis had been satisfactorily obtained in all cases with the use of a pressure pack for 30 minutes. Routine dental extractions can be safely performed in patients on long-term antiplatelet medication, with no interruption or alteration of their medication. Such patients do not have an increased risk of prolonged or excessive postoperative bleeding.

Effect of subcutaneous administration of dalteparin on lupus anticoagulant assays

Introduction Treatment with unfractionated heparin (UH) is known to affect screening tests for lupus anticoagulant (LA). False positive test results are common because confirmatory steps lack sufficient specificity to distinguish between LA and the presence of heparin. In this study, we wanted to see if therapeutic levels of low-molecular weight heparin (LMWH) may cause false positive tests for LA or alter the LA test results in LA-positive patients. We also wanted to evaluate the need to include heparin-neutralizing agents in the reagents. Materials and methods Six healthy subjects without LA and six LA-positive patients were given 100 IU/kg dalteparin subcutaneously (s.c.). Samples for three in-house and two commercially available LA tests were taken before and 4 h after the injection. LA test results were calculated as normalized screening/confirm ratios or as recommended by the manufacturers. Results With both healthy subjects and LA patients, only small and clinically unimportant differences in mean clotting times and final test results were seen 4 h after subcutaneous dalteparin injections, at anti-FXa activities within the therapeutic range. Conclusions Our study with dalteparin suggests that LMWH therapy with plasma concentrations within the therapeutic range does not cause false positive tests for LA when normalized screening/confirm ratios are applied; nor do test results for LA-positive patients seem to be significantly altered. Heparin-neutralizing agents did not influence test performance.

103 Autologous Thrombin: Continuing Studies

Background: As many as 500,000 patients are exposed to bovine thrombin (BT) annually. This is increasing with the use of autologous platelet gel (PG) in surgery and in the treatment of chronic wounds (diabetic ulcers). The development of anti‐phospholipid antibodies after exposure to bovine thrombin is of concern. We compared the kinetics of growth factor release by autologous thrombin (AT) to BT. Methods: Whole blood is incubated with 95% ethanol and then centrifuged in the SmartPReP™2 system (Harvest Technologies, Plymouth, MA) simultaneously with the preparation of a platelet concentrate (PC). The supernatant containing thrombin activity is collected. Clotting studies were done. The kinetics of growth factor release was determined by collecting the supernatant from the PG at 2 hours and daily thereafter for 7 days. The PC’s were prepared in two concentrations by adjusting the volumes to 10 mL and 7 mL. The activation of the PC was done at a 10:1 ratio (PC:BT) at 7 mL and 10 mL and 3:1 and 5:1 (PC:AT) at 7 mL. Results: Following a 45 minute incubation period the mean clotting time of PC by AT at a 3:1 ratio was 14.3 sec and at a 5:1 ratio was 22 sec (n = 13). Thrombin activity remained stable for 6 hours. Baseline PDGF‐AB levels with AT was 50 ng/mL with a maximum of 106 ng/mL. BT using a 10 mL PC had a baseline level of 100 ng/mL with no increase over time. TGF‐B1 studies demonstrated an immediate release of 98 ng/mL with a maximum level of 168 ng/mL for AT compared to BT with an initial level of 107 ng/mL and a maximum of 152 ng/ml. Conclusions: AT produces a sustained release of growth factors during a seven‐day period that is similar to BT at a 10:1 ratio. AT can be prepared simultaneously with the preparation of a PC.

In vivo estrogen bioactivities and in vitro estrogen receptor binding and transcriptional activities of anticoagulant synthetic 17β-aminoestrogens

Estrogenic activities of the two 17β-aminoestrogen (AE) derivatives, prolame and butolame, were studied upon coagulation, serum luteinizing hormone (LH) and uterine weight, including endometrial morphology in castrated female rats. We have also investigated the ability of these two compounds, as well as another AE pentolame, to activate transcription through the estrogen receptor α (ERα) and the estrogen receptor β (ERβ). Administration of prolame and butolame to castrated animals increased significantly ( P < 0.01) the mean clotting time when compared with that obtained in the group of control animals. Butolame was a more potent anticoagulant than prolame ( P < 0.01), as judged by their corresponding IC 50 (5.4 ± 0.65 and 66.6 ± 2.57 μg/animal, respectively). In contrast, estradiol significantly shortened blood clotting times ( P < 0.005). Both prolame and butolame caused a significant inhibition of serum LH levels (EC 50 8.10 ± 0.79 and 17 ± 64 μg/animal, respectively), and restored castration-induced reduction in uterine weight of ovariectomized rats (EC 50 4.14 ± 1.57 and 17.0 ± 1.78 μg/animal, respectively). In terms of the effects of prolame, butolame and pentolame in transient transfection assays, all the three AE activated ER dependent reporter gene expression, however, only at high concentrations. Prolame had the highest activity followed by butolame and pentolame. Induction of transcription by these compounds was preferentially mediated through the ERα, especially in the case of pentolame where little, if any, activation occurred through the ERβ. None of the compounds showed antagonistic activities through either ER subtype. The overall data suggest that modifications in the structure and length of the amino-alcohol side-chain at C-17 might have an impact on the affinity and estrogenic intrinsic properties of AE at the level of diverse target tissues.

Plasma sialidase activity in acute myocardial infarction

Increased plasma sialidase activity was demonstrated in 25 patients with acute myocardial infarction. The mean plasma sialic lcid level after incubation with substrate neuramin lactose was 0.060 ± 0.003 (SEM) μmol/sample compared with 0.017 ± 0.003 μmol/sample in 24 patients without infarction ( p < 0.001). The erythrocyte endothelial cell adhesion index was highest when both cell types were sialidase treated (0.35 vs control 0.00 [ p < 0.05]). When sialidase (50 U/ml) was added to whole blood in plastic tubes, the mean clotting time was 7.17 ± 1.0 (SD) minutes (n = 8) compared with a mean control of 15.45 ± 3.9 minutes ( p < 0.01). Sialidase had no effect on prothrombin or partial thromboplastin times. The above studies suggest that increased plasma sialidase activity in patients with acute myocardial infarction may be associated with clumps of desialylated erythrocytes that may alter flow in the microcirculation.

Revised standardization method of the one-stage prothrombin time.

The revised ratio method which is recommended for standardization of the one-stage prothrombin time, presumes that identical regression lines exist between clotting times of abnormal and normal plasma. A modification is proposed which corrects for possible deviation of the mean clotting time of normal plasma from the regression line between clotting times of abnormal plasma. The modified revised ration method is theoretically preferable and does not need the restrictions in calibration which are recommended for the revised ratio method.

Human alveolar macrophages synthesize factor VII in vitro. Possible role in interstitial lung disease.

Both fibrin and tissue macrophages are prominent in the histopathology of chronic inflammatory pulmonary disease. We therefore examined the procoagulant activity of freshly lavaged human alveolar macrophages in vitro. Intact macrophages (5 X 10(5) cells) from 13 healthy volunteers promoted clotting of whole plasma in a mean of 65 s. Macrophage procoagulant activity was at least partially independent of exogenous Factor VII as judged by a mean clotting time of 99 s in Factor VII-deficient plasma and by neutralization of procoagulant activity by an antibody to Factor VII. Immunoprecipitation of extracts of macrophages metabolically labeled with [35S]methionine by Factor VII antibody and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a labeled protein consistent in size with the known molecular weight of blood Factor VII, 48,000. The addition of 50 micrograms of unlabeled, purified Factor VII blocked recovery of the 48,000-mol wt protein. In addition, supernatants of cultured macrophages from six normal volunteers had Factor X-activating activity that was suppressed an average of 71% after culture in the presence of 50 microM coumadin or entirely by the Factor VII antibody indicating that Factor VII synthesized by the cell was biologically active. Endotoxin in vitro induced increases in cellular tissue factor but had no consistent effect on macrophage Factor VII activity. We also examined the tissue factor and Factor VII activities of freshly lavaged alveolar cells from nine subjects with clinical and/or histologic evidence of sarcoidosis. Four of the nine subjects expressed increased tissue factor and seven of nine had increased Factor VII activity over the normal range (P less than 0.01). We estimate the mean Factor VII associated with the cells of sarcoid patients to be 4.7 ng/10(6) cells (range 0.4-20) as compared to a mean of 0.74 ng/10(6) cells (range 0.2-2) for that of normal subjects. Along with previous data showing synthesis of plasminogen activator, these findings indicate that human alveolar macrophages normally synthesize and express measurable amounts of the initial enzymes of proteolytic reactions regulating both fibrin deposition and fibrin resorption. Abnormalities in Factor VII activity in a small group of patients with sarcoidosis raise the possibility that modulation of fibrin turnover by macrophages may contribute to the pathology of this and perhaps other interstitial lung diseases.

Comparative hematology: Studies on class aves, domestic turkey ( meleagris gallopavo)

1. 1. The mean clotting time (C.T.) for turkey blood was 71.0 min in glass and 83.0 min in siliconized tubes. Accelerated C.T.s with rabbit brain or partial reagent were over 60 sec but with turkey brain or RVV they were less than 20 sec. 2. 2. Fibrinogen averaged 450 mg/dl. Coagulation factors, assayed in human systems, were very low with the exception of XIII and VIII (0.57 U/ml). 3. 3. Thrombocytes aggregated during clotting and with collagen but not with ADP. Thrombocytes contained microtubules, an open canalicular system, a large nucleus but no α granules or dense bodies. 4. 4. Hemostasis appeared dependent on an extrinsic pathway.

Screening and evaluation of materials for thromboresistance.

AbstractTwo methods are described for evaluating the thromboresistance of materials available as tubing. In the first method lengths of the tubing containing canine blood were incubated at 37°C, opened at timed intervals and examined for clots. For all materials tested, mean clotting times ranged from 5.2 min for glass to &gt; 300 min for silicone rubber treated with GBH, TDMAC‐heparin or TDMAN‐heparin. For most materials, in vitro clotting times ranged from 20 to 60 min. Further testing in vitro indicated that heparin was more firmly bound to silicone rubber as TDMAC‐heparin than as GBH. In the second method, short segments of tubing were surgically placed within the femoral veins of anesthetized dogs, examined for patency after 2 hr, removed and examined for thrombi. None of the materials tested appeared completely thromboresistant by the 2 hr in vitro method. Tubes of glass or unheparinized materials were usually completely occluded at the end of the test; silicone rubber treated with TDMAC‐heparin apperared relatively thromboresistant. Results of in vitro and in vivo testing were generally in good agreement and were consistent with the known thromboresistant properties of materials.

An epidemiological study of varicose veins in Indian railroad workers from the South and North of India, with special reference to the causation and prevention of varicose veins.

A survey to determine the prevalence of varicose veins among railwaymen of identical socio-economic status and doing identical work of sweepers in the North and South of India showed that the overall prevalence was significantly higher among South Indian sweepers (25.08 per cent) than among North Indian sweepers (6.8 per cent). The survey included 323 men from Madras in the South and 354 men from Ajmer in the North of India. While constipation, body weight, smoking, posture and tight undergarments do not appear to contribute to the causation of varicose veins, the role of heredity could not be examined in this study. Big differences were present in blood-clotting time and in clot-lysis between these two populations, the respective values for clotting time being 6 minutes in South Indians and 8 minutes 40 seconds for North Indians (P ⋞ 0.001), and for clot-lysis being 3.7 per cent in South Indians and 25 per cent in North Indians. In order to test the cause-and-effect relationship of these haematological indices 24 North Indians and 81 South Indians with varicose veins were compared with 24 healthy North Indian controls and 81 South Indian controls with no varicose veins. Clot-lysis was markedly higher in those with no varicose veins than in those with varicose veins, but the mean blood clotting times were not significantly different between these two. Since such differences are known to be diet related, this study suggests that, in the prevalence of varicose veins, patterns of diet and eating may play an important part. Therefore, there would seem to be hope that this disease may be prevented.

Blood coagulation and anticoagulant mechanisms in the turtle Pseudemys elegans

1. 1. Three groups of control (warm) and three groups of experimental (cold-torpid) turtles ( Pseudemys elegans) were subjected to room temperature (21–22°C) and to low temperature (2–4°C) respectively, from 1 to 6 months. Blood coagulation was studied utilizing such standard tests as the prothrombin clotting time of plasma and the coagulation time of whole blood. An anticoagulant substance with the metachromatic staining properties of a mucopolysaccharide was extracted from plasma and an attempt was made to correlate the amount of this substance with mast cell populations of liver and duodenal tissues. 2. 2. The prothrombin clotting time of the warm and cold-torpid groups varied considerably, but the mean clotting times for the two groups were essentially identical. An absence of whole blood coagulation was noted in many animals from both warm and cold environments. It was concluded that neither the warm nor the cold-torpid animals had an apparent fibrinogen deficiency, and that the variation in the clotting time was possibly due both to other factors of the clotting process and to seasonal or other factors independent of the experimental environment. 3. 3. Mean yields for the extracted anticoagulant were 2·04 and 6·67 mg/100 ml plasma for the three groups of warm and the three groups of cold-torpid animals respectively. The yield of the anticoagulant increased with length of exposure to the experimental environment. 4. 4. No correlation was found between the quantity of the anticoagulant substance and the number of tissue mast cells in liver and duodenal tissues from warm or cold-torpid animals. Neither was correlation found between the prothrombin clotting time and the anticoagulant yield nor between the coagulation of whole blood and the anticoagulant yield.

Potencies of Vitamin Ki and of 2-Methyl-1, 4-Naphthoquinone

In a previous investigation1 we have confirmed the report of the marked antihemorrhagic activity of 2-methyl-1,4-naphthoquinone.2 A careful comparison of the potencies of this compound and of pure vitamin K1 by our 18-hour procedure3 showed that the latter is approximately one-half as active as the former. In view of this observation and the report that by the 6-hour procedure vitamin Ki is only 1/30 as potent as 2-methyl-1,4-naphthoquinone,4 it seems desirable to publish the results that we have obtained in a comparison of the potencies of the two compounds by the 6-hour observation period.Experimental. In our experiments, we have used for the evaluation of the response of the chicks: (1) the percentage of chicks showing a clotting time of less than 10 minutes;3 (2) the mean clotting time; (3) the mean prothrombin time.5 Following Ansbacher's suggestion, a solution of the compound in cod liver oil was administered and the blood drawn 6 hours later for the evaluation of the reaction. Each assay included t...