Another method to prevent venous thrombosis in microsurgery: an in situ venous catheter

Free-flap failure is in the order of 4 to 10 percent. Heparin is more effective at preventing venous thrombosis than arterial thrombosis. This study was undertaken to investigate the efficacy of delivering heparin at a high dose locally but low dose systemically (heparin infusion via a catheter placed proximal to the venous anastomosis) to prevent venous thrombosis in microsurgery. A model of venous thrombosis was first established by a venous inversion graft in the rat femoral vein (this was performed in seven animals and resulted in 100 percent thrombosis). Saline and heparin were delivered proximal to the inverted vein graft to assess the effect of each in preventing venous thrombosis. Flow/patency distal to the inverted vein graft was assessed by observation under the microscope, the milk test, and rate of flow (flowmeter). Saline infused via a catheter proximal to the venous inversion graft resulted in 100 percent thrombosis in 10 animals. Heparin (100 U/ml at 2 to 3 ml/hour) infused through a catheter for 2 hours proximal to the anastomosis resulted in flow in all 10 animals during the infusion. Blood was also taken before beginning the procedure (control) and after the heparin infusion distal to the anastomosis (local partial thromboplastin time) as well as in the contralateral femoral vein (systemic). The control for all animals that received heparin was <3 minutes. The systemic partial thromboplastin time after heparin infusion was <3 minutes in seven animals, 3.3 minutes in two animals, and >7 minutes in one animal. The local partial thromboplastin time distal to the inverted vein graft was >10 minutes in nine animals and 3.7 minutes in one animal. The study also had a clinical component, in which a catheter was placed in a vein of the free flap, and heparin was infused over 5 days. This technique has been used in 83 consecutive free flaps. In three recent free flaps performed on the limbs, the local partial thromboplastin time (close to the anastomosis) was raised but the systemic time was normal. This technique offers a method in preventing venous thrombosis in microsurgery. It is simple to implement and is not associated with the systemic complications of heparin.     

The effect of heparin and thromboplastin on the half-life of 125I-protein C in the blood flow of rats]

The influence of heparin and thromboplastin on the halflife of 125I-protein C in rat blood was under investigation. It was found that t1/2 of protein C was of 2.3 h. The intravenous administration of heparin resulted in the prolongation of t1/2 to 6.5 h, that could be explained by inhibition of thrombin generation. Upon the 40-min infusion of thromboplastin the rate of 125I-protein C decay in blood enhanced. That could be explained by the generation of the endogenous thrombin and participation of thrombomodulin in the protein C activation as well as in the removal of the endogenous thrombin from blood.     

Combined deficiency of factor V and factor VIII. A report of another case.

A patient with combined factor V and factor VIII deficiency is presented. The bleeding manifestations were: easy bruising, post-traumatic bleeding, bleeding after tooth extractions. The main laboratory feature was a prolonged partial thromboplastin time which was corrected by the addition of adsorbed normal plasma but not by the addition of normal serum, hemophilia A plasma of another patient with combined factor V and factor VIII deficiency. The thromboplastin generation test was clearly abnormal and was corrected by the addition of adsorbed normal plasma but not by addition of normal serum. Prothrombin consumption was also defective. Prothrombin time was slightly prolonged too, Thrombin time, platelet and vascular tests were within normal limits and there was no hyperfibrinolysis. Factor VIII was 8% of normal, whereas factor V was 14% of normal. Factor VIII associated antigen was normal. All other clotting factors were within normal limits. The parents of the propositus were consanguineous (first cousins) but had normal factor V and factor VIII activity and normal factor VIII antigen. The same was true for other family members. The hereditary transmission of the condition appears autosomal recessive.     

Anticoagulant activities of oleanolic acid via inhibition of tissue factor expressions

Oleanolic acid (OA), a triterpenoid known for its anti-inflammatory and anti-cancer properties, is commonly present in several medicinal plants but its anticoagulant activities have not been studied. Here, the anticoagulant properties of OA were determined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrin polymerization as well as cell-based thrombin and activated factor X (FXa) generation activities. Data showed OA prolonged aPTT and PT significantly and inhibited thrombin catalyzed fibrin polymerization. In addition, OA inhibited the activities of thrombin and FXa and inhibited the generation of thrombin or FXa in human endothelial cells. OA also inhibited TNF-α-induced tissue factor expression on human endothelial cells. In accordance with these anticoagulant activities, OA showed an anticoagulant effect in vivo. These results indicate that OA possesses antithrombotic activities and suggest that daily consumption of a herb containing OA may be preventing thrombosis in pathological states.     

Caulobacter cresentus mutant defective in membrane phospholipid synthesis.

To study the relationship between phospholipid synthesis and organelle biogenesis in the dimorphic bacterium Caulobacter crescentus, auxotrophs have been isolated which require exogenous glycerol or glycerol 3-phosphate for growth when glucose is used as the carbon source. Upon glycerol deprivation, net phospholipid synthesis ceased immediately in a glycerol 3-phosphate auxotroph which was shown to have levels of biosynthetic sn-glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) activity 10 times lower than that of the wild type. In the absence of glycerol, the optical density of the culture continued to increase for the equivalent of one generation, although the cells did not divide. After the equivalent of one generation time, rapid cell death occurred. Cell death also occurred when phospholipid synthesis was inhibited by cerulenin. Although ribonucleic acid and protein syntheses continued at a reduced rate for the equivalent of one generation in mutant strains, a substantial decrease in the rate of deoxyribonucleic acid synthesis occurred immediately upon glycerol deprivation. Revertant strains had wild-type levels of glycerol 3-phosphate dehydrogenase activity and normal rates of phospholipid and macromolecular synthesis.     

Anti-thrombosis effect of diosgenin extract from Dioscorea zingiberensis C.H. Wright in vitro and in vivo

Thrombus formation in blood vessel plays an important role in the pathogenesis and progression of atherosclerosis and cardiovascular diseases. Extract of Dioscorea zingiberensis C.H. Wright (D. zingiberensis) is demonstrated to posses activities for curing cardiovascular diseases such as coronary heart disease and angina pectoris. However, there were few studies on anti-thrombosis activity of it. We investigated the anti-thrombosis effect of diosgenin from D. zingiberensis (Dio) in vitro and in vivo on inferior vena cava ligation thrombosis rat model and pulmonary thrombosis mice model. We evaluated the protective effect of Dio by measuring the platelet aggregation, activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and the venous thrombosis in rats and the bleeding time, clotting time and protection rate in mice. Results showed that Dio inhibited platelet aggregation, thrombosis and prolonged APTT, PT and TT in rats in a dose-dependent manner. They also prolonged the bleeding time, clotting time and increased protection rate in mice in a dose-dependent manner. Taken together, these findings suggested that Dio which contained 95% diosgenin had anti-thrombosis activity. Dio executives the anti-thrombosis activity through improving the anticoagulation function, inhibiting platelet aggregation and thrombosis.     

Diphtheria toxin resistance in human fibroblast cell strains from normal and cancer-prone individuals

Mutants resistant to diphtheria toxin (Dip^r) have been selected from a variety of human fibroblast cell strains derived from both normal subjects and individuals with known genetic predisposition to cancer such as xeroderma pigmentosum, Fanconi anemia and Bloom's syndrome. Treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) led to a marked increase in the frequency of Dip^r mutants in various cell strains. The increase in the frequency of Dip^r mutants occurred in a linear dose-dependent manner in response to MNNG and ethyl methanesulfonate, in one of the cell strains examined. The rate of muation to diphtheria toxin as determined by fluctuation analysis was very similar in various cell strains (1–3 × 10^?7 mutations/cell/generation), except for the strain GM1492 (8.8 × 10^?7 mutations/cell/generation) which is derived from a Bloom syndrome patient.     

The report of an Italian family with heterozygous protein C deficiency

A heterozygote protein C deficit was found in 4 members of the same family. The propositus is a 40 year old male with a clear thrombotic tendency. This included repeated thrombophlebitis of the right leg, and one episode of pulmonary embolism. Arterial thrombosis was not noted. The anticoagulant therapy undertaken by the patient appears to be of some benefit in the sense that no recurrence of thrombotic manifestations occurred. One brother and two nephews of the propositus, even though asymptomatic showed reduced levels of Protein C both as activity and antigen. The parallel reduction of Protein C activity and antigen points towards a "true" deficit of Protein C. The normal, although reduced, pattern in the crossed immunoelectrophoresis supplies further confirmation to this interpretation.     

The reactivity of the anticoagulatory system in sympathectomized animals under stress conditions due to acoustic trauma]

In rats of kind of Kruschinskij-Molodkina the number of cells in the stellate ganglion was diminished to 0.5% of the norm after eliminating the sympathetic tonus by means of guanethidine. These animals died of cardiac thrombosis during stress situations. This thrombosis cannot be explained by adrenalin, corticosteroids or thromboplastin spreading in the blood stream. A factor, the nature of which has still to be explained, may be assumed to be responsible for thrombosis to develop during stress situations in animals whose sympathetic tonus has been eliminated by guanethidine.     

Deletion of P1 arginine in a novel antithrombin variant (antithrombin London) abolishes inhibitory activity but enhances heparin affinity and is associated with early onset thrombosis

A novel variant of antithrombin, the major serpin inhibitor of coagulation proteases, has been identified in a patient with early onset thrombosis and abnormal plasma antithrombin activity. Sequencing of the antithrombin genes of the patient revealed that one of the two alleles was abnormal due to an in-frame deletion of the codon for the P1 arginine residue. The abnormal antithrombin was separated from the normal inhibitor by complexing the latter with thrombin followed by heparin-agarose affinity chromatography. The purified variant, antithrombin London, was completely inactive as a thrombin or factor Xa inhibitor even after heparin activation. Surprisingly, the variant bound heparin with a K(D) reflecting an approximately 10-fold greater affinity than the normal inhibitor. Stopped-flow kinetic analysis showed that this was almost entirely due to a more favorable conformational activation of the variant than the normal inhibitor, as reflected by a decreased rate constant for reversal of the activation. Consistent with its higher than normal heparin affinity, the inactive antithrombin variant was a potent competitive antagonist of the heparin-catalyzed reaction of normal antithrombin with thrombin but did not affect the uncatalyzed reaction. These results suggest that deletion of the antithrombin P1 residue partially activates the serpin by inducing strain in the reactive center loop, which destabilizes the native loop-buried state and favors the activated loop-exposed state with high heparin affinity. The unusually severe thrombosis associated with the heterozygous mutation may be explained by the ability of antithrombin London to bind endogenous heparan sulfate or heparin molecules with high affinity and to thereby block activation of the normal inhibitor.     

Prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin administration

The possibility of prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin devoid of proteolytic activity and capable of stimulating the reaction of anticoagulation system was studied. The injection of lethal thromboplastin dose was shown to produce a sharp increase in soluble fibrin blood content, total disappearance of fibrinolytic activity and intravascular blood coagulation. The animals died of thrombosis in 90% of cases. It was established that the injection of lethal thromboplastin dose 5 min after DIP-alpha-thrombin injection caused a 13% lethality from thrombosis. No reliable changes in fibrinolytic activity and soluble fibrin content were observed. A significant increase in thrombin and recalcification time was recorded. It is suggested that DIP-alpha-thrombin prevents intravascular blood coagulation induced by lethal thromboplastin dose due to mobilization of the reserve capacities of neuro-humoral anticoagulation system.     

Antiphospholipid antibodies in young myocardial infarction patients

Myocardial infarction (MI) is a multi-factorial disease which claims many young lives. There are very few Indian studies that have investigated antiphospholipid antibodies (APLs) in MI patients. APLs have been implicated in arterial thrombosis including premature coronary artery and cerebrovascular thrombosis. In the present study, the prevalence of two clinically significant APLs--anticardiolipin antibody (ACA) and lupus anticoagulants (LA) in young MI patients was studied and compared with age- and sex-matched controls. Fifty healthy blood donors and 40 young MI patients (less than 45 yrs) diagnosed according to the American Heart Association guidelines were recruited for the study. The criteria for diagnosis were presence of atleast two of three classical findings including: clinical symptoms, diagnostic ECG, and presence of one or more cardiac biomarkers out of raised CK-MB isoform and T-troponin on serial measurement. LA and ACA were tested by lupus-sensitive activated partial thromboplastin time (aPTT) and ELISA respectively. Elevation of ACA was observed in 9 patients, while 6 were positive for LA. ACA of IgG isotype was detected in 8 patients. One patient had LA and raised ACA of IgG and IgM isotypes. Antiphospholipid antibodies were found to be significantly associated with MI in young patients, when considered together (p < 0.05) and in coronary thrombosis, mild elevation of ACA may be considered significant.     

Antithrombotic effect of tissue and plasma type angiotensin converting enzyme inhibitors in experimental thrombosis in rats.

This study compared the antithrombotic effect of plasma angiotensin converting enzyme inhibitors (ACE-Is): captopril (CAP), enalapril (ENA) and tissue ACE-Is: perindopril (PER), quinapril (QUIN) in experimental venous and arterial thrombosis. Normotensive Wistar rats were treated p.o. with CAP (75 mg/kg), ENA (20 mg/kg), PER (2 mg/kg) and QUIN (3 mg/kg) for 10 days. The influence of ACE-Is on coagulation and fibrinolytic systems as well as platelet function was evaluated. The hypotensive effect of ACE-Is was equal in all groups. QUIN maintained the final carotid blood flow at the highest value in comparison to PER and plasma ACE-Is. The arterial thrombus weight was reduced in PER and QUIN groups while venous thrombus weight was also reduced after CAP. Tissue and plasma ACE-Is caused the inhibition of platelet adhesion and aggregation. A reduction of fibrin generation, prolongation of prothrombin time (PT), activated partial thromboplastin time (APTT) and shortening of euglobulin clot lysis time (ECLT) were observed after PER and QUIN treatment. In conclusion, given in equipotent hypotensive doses, tissue ACE-Is exerted more pronounced antithrombotic effect than plasma ACE-Is in experimental thrombosis. The differences between tissue and plasma ACE-Is in terms of their more pronounced inhibition of experimental thrombosis may be related to the intensified activation of fibrinolysis and inhibition of coagulation.     

The changing outlook in coronary disease

The statistical prognosis for patients who survive a first attack of coronary thrombosis, as regards both life expectancy and ability to return to normal activity, has been greatly improved in recent years. In the light of advances in understanding of the physiology of the heart and improvements in therapeutic methods, physicians must reevaluate ideas of what a patient should be permitted to do after recovery from an initial attack. Often a return to normal pursuits may be better for the patient than drastic restriction of activity, particularly because of the psychological and emotional effects of invalidism. In deciding what advice to give on this score, the physician should consider in each case not only the actual amount of coronary circulation but such factors as the patient's temperament, type of occupation and economic status. The goal should be to guide each patient back to usefulness within the limits of his cardiac reserve.     

Tissue plasminogen activator inhibitor in patients with systemic lupus erythematosus and thrombosis.

OBJECTIVE--To examine the relations among tissue plasminogen activator antigen, plasminogen activator inhibitor, the lupus anticoagulant, and anticardiolipin antibodies in patients with systemic lupus erythematosus. DESIGN--Prospective study of blood samples (a) from selected patients with systemic lupus erythematosus whose disease was and was not complicated by a history of thrombosis or recurrent abortions, or both, and (b) from a series of healthy controls with a similar age and sex distribution. SETTING--University based medical clinic. SUBJECTS--23 Patients with definite systemic lupus erythematosus (American Rheumatism Association criteria), of whom 11 (eight women) aged 26-51 had a history of thrombosis or recurrent abortions, or both, and 12 (10 women) aged 23-53 had no such history. 15 Healthy subjects (10 women) aged 25-58 served as controls. MAIN OUTCOME MEASURES--Tissue plasminogen activator concentrations, plasminogen activator inhibitor activities, detection of the lupus anticoagulant, and values of anticardiolipin antibodies in the two groups of patients and in the patients with a history of thrombosis or abortions compared with controls. Other measurements included concentrations of proteins that are known to change during the acute phase of systemic lupus erythematosus--namely, fibrinogen, C3 and C4, and C reactive protein. RESULTS--Patients with a history of thrombosis or abortions, or both, had significantly higher values of tissue plasminogen activator and plasminogen activator inhibitor than patients with no such history. A significant correlation between tissue plasminogen activator and plasminogen activator inhibitor (r = 0.80) was found only in the patients with a history of complications of their disease. The lupus anticoagulant was detected in six of the 11 patients with a history of thrombosis or abortions when tested by measuring the activated partial thromboplastin time but was found in all 11 patients when tested by measuring the diluted activated partial thromboplastin time. Nine of these 11 patients had raised values of anticardiolipin antibodies. The findings showed no relation to the activity of the disease. CONCLUSIONS--A significant correlation between tissue plasminogen activator concentrations and plasminogen activator inhibitor activities was found only in patients whose systemic lupus erythematosus was complicated by a history of thrombosis or recurrent abortions. The findings show that these patients have raised plasminogen activator inhibitor activities, and the frequent association between these raised activities and the presence of the lupus anticoagulant suggests that the two may be linked.     

Results of ultra-high streptokinase (Awelysin) doses in patients with thrombophlebitis

The results of 6-hour fibrinolysis with ultra-high streptokinase doses in 30 patients with peripheral venous thrombosis were presented. In 33.3% of the patients complete, and in 40% partial repatency was attained, while no repatency occurred in 20%. Deterioration was observed in two patients. The most beneficial effects were seen in postoperative and posttraumatic thrombosis. Bleeding complications did not occur. In one female patient treatment was discontinued due to bronchospasm.     

Prevention of Early Postoperative Deep Vein Thrombosis by Intermittent Compression of the Leg during Surgery

A clinical trial is described in which the effect of intermittent compression of the lower limb during surgery on the incidence of early postoperative deep vein thrombosis was assessed. Deep vein thromboses were diagnosed by the ¹²⁵I-fibrinogen uptake test. Peroperative intermittent compression was achieved by means of an inflatable plastic splint coupled to a pneumatic controller. By compressing only one leg of each patient, each patient acted as his own control.With a sequential statistical analysis, 39 patients were required to pass the 5% level of significance. Eleven thrombi were detected in the control (uncompressed) legs and two occurred in the compressed legs; one of the latter was bilateral. The investigation shows that increasing the pulsatility of the venous flow in the leg is a potent prophylactic against postoperative deep vein thrombosis.     

Effect of ethyloestrenol on fibrinolysis in the vessel wall.

Forty-nine patients with decreased fibrinolytic activity in the vessel walls or a decreased release mechanism, or both, were treated with ethyloestrenol for three to 17 months. Forty-five of the patients had had recurrent, phlebographically verified, deep venous thrombosis (DVT) and four had arterial thrombosis. Ethyloestrenol 8 mg/day was given to 31 patients and 4 mg/day was given to 12. The remaining six patients had been treated with a combination of phenformin and ethloestrenol. The phenformin was withdrawn but they were kept on ethyloestrenol 8 mg/day. Another 15 patients with a normal fibrinolytic system--four with recurrent DVT and 11 with severe arteriosclerosis--were given ethyloestrenol 8 mg/day. The spontaneous fibrinolytic activity, local fibrinolytic activity during standardised venous occlusion of the arms, and fibrinolytic activity of the vessel walls increased significantly after treatment with ethyloestrenol 8 mg/day for three months. No further increase occurred after three months, and ethyloestrenol 4 mg/day had no effect. No values rose significantly in the patients with a normal fibrinolytic system. One patient suffered a recurrence within three months of treatment, before the fibrinolytic system became normal. In one patient the fibrinolytic defect reappeared after 10 months in spite of continued treatment. Two of the three women of fertile age developed irregular cycles and intermenstrual bleeding, which disappeared when the treatment was withdrawn. No other side effects were observed.     

Microplate coagulation assays.

This report describes the development of microplate-based blood coagulation assays. The assays require a kinetic microplate reader to follow changes in absorbance at 405 nm caused by the coagulating plasma. Procedures for performing prothrombin time and activated partial thromboplastin time tests are described with intra- and inter-assay variability of a few percentage points. The prothrombin time of normal plasma was 64.5 +/- 3.6 s, and the activated partial thromboplastin time was 69.8 +/- 3.2 s. Clotting times were prolonged when normal plasma was mixed with plasmas deficient in particular coagulation factors, as expected. These assays take advantage of the microplate format (small sample size and multiple simultaneous assays) and can be customized for specific purposes, such as quantifying purified factor IX or assessing protein C activity in plasma.     

THE CHANGING OUTLOOK IN CORONARY DISEASE

The statistical prognosis for patients who survive a first attack of coronary thrombosis, as regards both life expectancy and ability to return to normal activity, has been greatly improved in recent years. In the light of advances in understanding of the physiology of the heart and improvements in therapeutic methods, physicians must reevaluate ideas of what a patient should be permitted to do after recovery from an initial attack. Often a return to normal pursuits may be better for the patient than drastic restriction of activity, particularly because of the psychological and emotional effects of invalidism.In deciding what advice to give on this score, the physician should consider in each case not only the actual amount of coronary circulation but such factors as the patient''s temperament, type of occupation and economic status. The goal should be to guide each patient back to usefulness within the limits of his cardiac reserve.