摘要:

A unique blood coagulation factor X variant has been identified in a family with a history of bleeding. Plasma from affected family members had prolonged prothrombin times and activated partial thromboplastin times, low to below normal factor X coagulant activity, and normal factor X antigen levels. Sequencing of DNA from the propositus revealed a single G to A substitution in one allele of factor X at base 964 resulting in an amino acid substitution of Asn for Asp at residue 282. This residue corresponds with the active site Asp102of chymotrypsin. The substitution eliminates aTaqI restriction site and provided the basis for a screening assay to detect the mutation in polymerase chain reaction (PCR) amplified factor X exon VIII DNA. Fourteen additional family members were identified as having the mutation at base 964. Plasma factor X purified from the proposita using an anti-factor X monoclonal antibody immunoadsorbent exhibited an approximately 50% decrease in specific activity compared with factor X purified from a normal individual in a similar manner. Bleeding in family members with the mutation, termed factor X Stockton, appears to be due to disruption of normal hemostasis by the presence in plasma of circulating abnormal factor X. Factor X Stockton is the first naturally occurring substitution at the active site Asp of a serine protease and underscores the importance of this amino acid residue in factor Xa coagulant activity.

ISSN:0957-5235    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

We report the results of protein C gene (PROC) analysis in a Spanish family with hereditary PC deficiency characterized by the presence of three siblings with PC anticoagulant activity levels clearly below 50% of normal and PC antigen and amidolytic activities between 50 and 75% of normal. Their parents are first cousins and have PC levels between 50 and 80% of normal. Sequence analysis of the whole coding sequence of thePROCgene revealed that the three siblings are double homozygotes for a G to A transition at nucleotide 3203 that replaces arginine 87 by histidine (R87H) and for another G to A transition at nucleotide 7054, in intron 7 (7054G → A). Both parents and one sister were found to be double heterozygotes for these two mutations. Screening for the intronic mutation in a control group and RT-PCR cDNA studies from ectopically transcribed mRNA indicated that 7054G→A is most likely a rare but neutral DNA variant. These results and the fact that heterozygosity for the missense R87H mutation has also been found associated with a slightly decreased PC anticoagulant activity in another Spanish family, lead us to conclude that homozygosity for R87H is responsible for the PC deficient phenotype in these three siblings.

ISSN:0957-5235    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Inhibition of thrombin generation by heparin and low-molecular-weight (LMW) heparins is an important parameter which relates to their anticoagulant actionsin vivo.Previous studies in our laboratory used a clotting assay for assessment of thrombin generation but other published studies have used a chromogenic method. We have therefore measured the inhibition of thrombin generation by unfractionated heparin (UFH) and LMW heparins by a modified chromogenic method using microtitre plates and compared the results with the clotting method. The degree of inhibition of thrombin generation when calculated from both peak thrombin concentrations and areas under the curve in the chromogenic assay was the same. When the activities of each heparin were expressed as EC80, i.e. concentrations required for 80% inhibition of thrombin generation, the EC80were higher in the chromogenic assay than in the clotting system. However when the potencies of the LMW heparins were expressed as percentages of that of the UFH standard by parallel line analysis, the differences between clotting and chromogenic assay results were small and not statistically significant (P> 0.05). This study demonstrates the feasibility of measuring inhibition of thrombin generation by a modified chromogenic method using microtitre plates, and shows that the results with this method are similar to those obtained with the clotting method.

ISSN:0957-5235    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Increasing dilutions of lupus anticoagulant (LA) plasmas from twelve patients were used to directly compare the sensitivity of four tests for LA. The tests evaluated were the modified Bell and Alton activated partial thromboplastin time (APTT), an APTT using a commercially prepared partial thromboplastin (Platelin LS APTT), a modified dilute Russell's viper venom time (DRWT), and a modified kaolin clotting time (KCT). LAs were detected in all twelve plasmas by each of three tests and eleven of twelve plasmas in a fourth test when undiluted patient plasma was used. Repeating the tests after diluting the LA plasmas with normal platelet-free plasma (PFP) showed that the KCT was the most sensitive test for LA, detecting eleven of twelve LAs at a dilution of 10% patient plasma and ten of twelve LAs at a dilution of 5% patient plasma. The modified Bell and Alton APTT and the modified DRWT had similar sensitivities at a patient plasma concentration of 10%, detecting seven of twelve and eight of twelve LAs, respectively. The Platelin LS APTT detected only four of twelve LAs at a patient plasma concentration of 10%. Our results indicate that the modified KCT is a sensitive method for the detection of LAs. The modified Bell and Alton APTT and the DRWT were less sensitive.

ISSN:0957-5235    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

The success of current thrombolytic strategies is undermined by ongoing thrombin activity, but it is uncertain whether prevention of thrombin generation or direct thrombin antagonism is effective in achieving more optimal thrombolysis. To address this question, 24 dogs with electrically induced coronary thrombus undergoing thrombolysis with tissue-type plasminogen activator (1 mg/kg) over 20 min, were given one of the following adjunctive regimens in a random fashion. Twelve dogs received saline, and served as the control group; a direct thrombin antagonist, hirudin, was given at a dose of 20 μg/kg/min for 90 min to six dogs, and a selective factor Xa inhibitor, tick anticoagulant peptide (TAP), was administered to six dogs at a dose of 30 μg/kg/min for 90 min. The time to reperfusion was similar in the saline and hirudin groups (34 ± 4vs37 ± 7 min;P=NS) but shorter in the TAP group (21 ± 4 min;P< 0.05). Coronary blood flow was restored to 100% of its baseline value for 7 ± 2 min in control dogs, and for 20 ± 6 min in the hirudin group (P< 0.05). In the TAP group, coronary blood flow was restored to 100% of its baseline value for more than 120 min in all dogs (P< 0.01vsothers treatments). Reocclusion occurred in 89% and 50% of dogs receiving saline and hirudin, respectively (P= NS), but in none of the TAP-treated dogs (P< 0.01). Plasma fibrinopeptide A (FpA) and thrombin-antithrombin III complex (TAT) levels were determined in all dogs as indicators of thrombin activation. In the saline group, FpA and TAT during reperfusion were 19 ± 2 ng/ml and 104 ± 24 ng/ml respectively (P< 0.02vsbaseline) indicating high thrombin activity. In contrast, during reperfusion in hirudin-treated dogs FpA and TAT remained similar to baseline (10 ± 3 ng/ml and 53 ± 4 ng/ml respectively; bothP< 0.05vssaline). Reperfusion in TAP-treated dogs did not alter FpA and TAT in plasma, which remained similar to baseline (9 ± 1 ng/ml and 39 ± 5 ng/ml respectively; bothP< 0.05 vs saline). Scanning electron microscopy of coronary arteries showed residual thrombi with intense platelet and fibrin deposition adherent to the deendothelialized surface of the vessels following saline and hirudin therapy. In contrast, TAP-treated arteries were characterized by the absence of fibrin and minimal platelet deposition. In conclusion, these hemodynamic, biochemical and morphologic data suggest that adjunctive treatment with a higher tier blockade of the coagulation cascade is superior to direct thrombin inhibition in maintaining coronary artery patency following thrombolysis in the experimental canine electrolytic model. These findings highlight the potential adverse effects of unchecked thrombin generation in the setting of thrombolytic therapy.

ISSN:0957-5235    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Low-molecular-weight (LMW)-dermatan sulfate (Desmin) with the mean molecular weight of 5600 Da has been obtained by limited depolymerization of natural dermatan sulfate. The pharmacokinetic and pharmacodynamic data of 100 and 200 mg were analyzed after intravenous injection and of 50,100 and 200 mg after subcutaneous injection on tissue factor pathway inhibitor (TFPI) antigen and activity, heparin cofactor (HC) II activity, HeptestTMcoagulation value, chromogenic S-2222 anti-factor Xa (aXa) assay, activated partial thromboplastin time (APTT), thrombin clotting time (TCT), plasminogen, tissue plasminogen activator activity (t-PA) and plasminogen activator inhibitor (PAI). After i.v. injection of 100 mg and 200 mg Desmin TFPI antigen and activity increased 2.2-and 2.7-fold, and returned to normal values within 60 and 90 min, respectively. Using the HC II assay the elimination half-lives (T1/2 el) increased from 1.9 h to 3.3 h with increasing doses of LMW-dermatan sulfate. T1/2 el were 4.3 and 6.9 h with the Heptest assay and 3.3 and 5.1 h with the aXa method, respectively. APTT, TCT and the fibrinolytic parameters were not modified by either dose of i.v. LMW-dermatan sulfate. After s.c. administration of 100 mg or 200 mg LMW-dermatan sulfate no increase of TFPI antigen or activity was detected. T1/2 el was 5.6 h using HC II method, 11.1 h using Heptest and 7.8 h with the aXa activity. The total clearance was about ten-fold higher when determined by the HC II method compared with Heptest and aXa method. The volume of distribution (VD) increased with increasing doses of s.c. LMW-dermatan sulfate and was highest with the HC II method. Intravenous administration of 100 mg protamine chloride 15 min after i.v. dosing of 100 mg LMW-dermatan sulfate did not modify TFPI, coagulation or fibrinolytic parameters. Further analysis of the complex mechanism of action has to include studies which should explain the low release of TFPI in relation to the antithrombotic effects of LMW-dermatan sulfate.

ISSN:0957-5235    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Arterial thrombosis in the rabbit was established as a novel model to evaluate the effects of antithrombotic agents. Endothelial injury was produced by applying electrical stimulation to the femoral artery. The process of primary endothelial injury, and subsequent platelet activation and fibrin formation were confirmed by electron microscopy. In this model, vessel occlusion occurred within 30 min after stimulation without changes in heart rate and blood pressure. Using this model, several agents were evaluated for their antithrombotic activities: aspirin (30 mg/kg, p.o.), ticlopidine (10–100 mg/kg, p.o.), heparin (300 unit/kg, i.v.), PPACK (10–33 μg/kg/min, i.v.), WEB-2347 (1 mg/kg, p.o.) and nicardipine (10 μg/kg, i.v.). Fifty per cent decrease in vessel temperature (Tp, assessed by a thermic probe, averaged 15.1 ± 1.2 (n= 11, p.o.) and 15.6 ± 1.9 min (n= 8, i.v.) in the vehicle groups, and this was significantly prolonged by aspirin (23.0 ± 2.6 min), ticlopidine at a dose of 100 mg/kg (24.6 ± 2.5 min), heparin (27.2 ± 2.8 min) and PPACK at a dose of 33 μg/kg (30.0 min). However, WEB-2347 and nicardipine were without effect. The effect of aprosulate, a new class of polyanion with anticoagulant activity, was further examined. Aprosulate (1–30 mg/kg, i.v.) inhibited thrombus formation in a dose-dependent manner. These results show that acute occlusive thrombus can be readily and reproducibly formed in the rabbit femoral artery and suggest that this thrombus formation depends on the activation of both platelets and blood coagulation. The merit of this model lies in its simplicity for evaluating the antithrombotic effects of antiplatelet and anticoagulant agents and is therefore expected to be extensively used in the future.

ISSN:0957-5235    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

A 43-year-old white female underwent orthotopic liver transplantation in 1992 for cirrhosis related to primary sclerosing cholangitis. Pre-transplantation protein S (PS) studies revealed a discrepancy between PS activity and free PS antigen consistent with type II PS deficiency. Since the presence of activated protein C (APC) resistance has been reported to interfere with PS activity assays resulting in an apparent type II PS deficiency, we retrospectively tested a pre-transplantation frozen plasma sample for APC resistance. The sample was found to have an abnormal APC resistance ratio (APCR-R) of 1.71. Follow up testing 2 1/2 years post-transplantation revealed correction of the APC resistance phenotype (normalization of the APCR-R to 2.79). Analysis of DNA extracted from lymphocytes revealed the patient to be heterozygous for the FV mutation associated with APC resistance (FV Leiden). Hereditary APC resistance was confirmed by family studies which revealed the presence of APC resistance and heterozygous FV Leiden in her son. Although the patient's post-transplantation plasma FV is normal, her platelet FV remains heterozygous for FV Leiden. To what extent, if any, platelet FV Leiden in the absence of plasma FV Leiden may contribute to a predisposition to thrombosis is unknown.

ISSN:0957-5235    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Inogatran (MW 439 Da), a new, selective, active site inhibitor of thrombin, was evaluated in three rat models of thrombosis. In the venous thrombosis model, inogatran dose-dependently inhibited thrombus formation with a > 80% antithrombotic effect at a plasma concentration of 0.45 μmol 1–1. In the arterial thrombosis model, inogatran dose-dependently inhibited thrombus formation, preserved vessel patency and the mean blood flow. Acetylsalicylic acid (ASA) potentiated the effects of low plasma concentrations of inogatran in the arterial thrombosis model. In the model of rt-PA-induced thrombolysis of a thrombus in the carotid artery, inogatran improved the patency time and the cumulative blood flow during the two hour thrombolysis period more than rt-PA alone. At high therapeutic plasma concentration of inogatran, there was only a moderate prolongation of bleeding time compared with the control value. It is concluded that inogatran is an effective antithrombotic agent both in the venous and arterial thrombosis models and also as adjuvant to rt-PA in the thrombolysis model.

ISSN:0957-5235    

出版商:OVID    

年代:1996

数据来源: OVID

摘要:

Production of 12-L-hydroxy-5,8,10-heptadecatrienoic acid (12-HHT) from platelets and bleeding times were studied in 32 males during acetylsalicylic acid (ASA) treatment and 1 and 2 weeks after withdrawal. All patients (age 42–77 years) had ASA treatment because of angina pectoris. The metabolite 12-HHT is formed in the same amount as the proaggregatory and vasoactive metabolite thromboxane A2. Initially the daily ASA dose was 75 mg (n= 15), 160 mg (n= 12) or 250–300 mg (n= 5). In all patients, median 12-HHT level increased from 40 to 240 g/750 × 106platelets (P< 0.001) 1 week after withdrawal of ASA, and four patients had abnormally high values. Median bleeding time decreased from 312 to 268 s (P= 0.003) in the 75 mg group and from 315 to 235 s in the 160 mg group (P= 0.01). Two weeks after withdrawal of ASA, median 12-HHT was 390 g/750 × 106platelets and eight patients (25%) had abnormally high values. One patient still had a prolonged bleeding time. Wide interindividual variations were observed in all groups. Our results indicate that rapid withdrawal of ASA, may cause abnormally high 12-HHT levels reflecting increase of thromboxane A2with possible hazardous effects in patients with cardiovascular disease.

ISSN:0957-5235    

出版商:OVID    

年代:1996

数据来源: OVID