Clinical relevance of the fibrinogen uptake test in patients undergoing elective general abdominal surgery—relation to major thromboembolism and mortality

Postoperative thromboembolic complications were evaluated in 2578 patients undergoing elective abdominal surgery, all receiving prophylaxis with low molecular weight heparin. A positive fibrinogen uptake test (FUT) developed in 217 patients (8.4%), while 37 patients (1.4%) had major thromboembolism (TE, defined as proximal deep vein thrombosis and/or pulmonary embolism, verified with phlebography, pulmonary scintigraphy or autopsy). In only 14% a positive FUT was associated with a major TE event. In 19% of the patients with major TE the FUT was negative. In multiple logistic regression the independent predictors for major TE were partially different from those for positive FUT. Thirty day mortality was 3.0%. There were significant associations between both positive FUT and major TE on one hand and mortality on the other (relative risks 2.4 and 5.8, respectively). FUT is not a good predictor of major TE. Both positive FUT and major TE indicate a significant risk of postoperative death.     

Long-term effects of ticlopidine on fibrinogen and haemorheology in patients with peripheral arterial disease

The effects of ticlopidine treatment (250 mg b.i.d. for 21 months) on fibrinogen and other rheological variables, as compared to placebo, were studied in 44 patients with intermittent claudication due to peripheral arterial occlusive disease. Blood samples were collected every 3 months during this double-blind, randomised placebo-controlled trial which lasted 21 months. Consistently lower values of fibrinogen, haematocrit and whole blood viscosity at high and low shear rate levels were found in the ticlopidine group; the intergroup differences were statistically significant at most but not all follow-up examinations. A significant time-related variance was observed in the ticlopidine group for the measured variables, also after correction for the variability found in the placebo group. Thus, the observed changes in the ticlopidine group are mainly treatment related. These effects on fibrinogen and haemorheology may contribute, besides the known antiplatelet activity of the drug, to the clinical improvement reported in a larger group of claudicants to which the present subset of patients belong.     

Studies on the haemostatic system in peripheral arterial disease I. Factor VIII, fibrinolytic activity, and changes induced by venous occlusion and nicotinic acid administration

Factor VIII coagulant activity (FVIII:C) and fibrinolytic activity (FA) were measured in a group of patients with peripheral arterial disease (PAD) and in a comparable control group.

The effects of two stimuli, venous occlusion (VO) and nicotinic acid administration (NA), were also investigated.

Significant or highly significant increases of both activities were found after VO and after NA in both groups. A linear correlation was seen between basal and post-stimulus values for FVIII:C, while only a logarithmic relation was present for FA in the same conditions. No correlation was observed between the two activities, either before or after stimuli. The response of FVIII:C to VO was correlated with that to NA, while no similar correlation was evident for FA.

No significant difference was recorded between patient and control group, both for FVIII:C and for FA, before and after the considered stimuli.     

Sphingomyelinase and cell-permeable ceramide analogs increase the release of plasminogen activator inhibitor-1 from cultured endothelial cells

We investigated the effect of exogenous staphylococcal sphingomyelinase (SMase) on the release of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) from cultured human umbilical vein endothelial cells (HUVEC). Addition of SMase (2 units/ml) to the culture medium induced an approx. 15-fold increase in the extracellular level of PAI-1 antigen at 3 h. No significant increase in the level of t-PA antigen was detected. Treatment of HUVEC with SMase (2 units/ml) for 3 h resulted in a significant decrease in the cellular sphingomyelin (SM) level, accompanied by a corresponding increase in the ceramide level. Cell-permeable ceramide analogs also enhanced the release of PAI-1 from cultured HUVEC in concentration- and time-dependent manners. A 6-fold increase in PAI-1 antigen level was observed after incubation for 3 h with 10 μM N-acetylsphingosine. Similar effect was noted as early as 2 h with 10 μM N-hexyanoylsphingosine. Addition of sphingosine failed to affect the release of PAI-1 from cultured HUVEC, indicating that the effects of ceramide analogs were independent of sphingosine generation. Pretreatment with cycloheximide or actinomycin D abated the response of HUVEC to N-acetylsphingosine in the increased levels of both extracellular and intracellular PAI-1. These results suggest that ceramide, generated via “SM cycle”, acts as a lipid mediator of PAI-1 release from vascular endothelial cells, and may contribute to a better understanding of the pathogenesis of the PAI-1-associated thrombotic disorders.     

In vitro effects of hyperlipoproteinemic sera on human endothelium: Inhibition of endothelial cell migration by familial hypercholesterolemic sera

Since hyperlipoproteinemia is associated with an increased risk of atherosclerosis we have evaluated the effects of sera of different hyperlipoproteinemic clinical patterns on human endothelial cells in vitro. Cultured human umbilical vein endothelial cells were treated with sera from 2 patients homozygous for familial hypercholesterolemia and 4 patients heterozygous for that disorder. Familial hypercholesterolemic sera inhibited endothelial cell migration by 50% during a 72 hour incubation (p <0.0001) compared to normal pooled human serum or single donor AB serum when measured by an agarose gel technique. The inhibition of migration was not observed when cells were treated with familial combined hyperlipidemic sera (4 patients) or familial hypertriglyceridemic sera (5 patients). Endothelial cell detachment in vitro was not induced by any of the classical patterns of hyperlipoproteinemic sera tested. The development of atherosclerosis in familial hypercholesterolemia may be in part related to an impairment of endothelial repair.     

Studies on the haemostatic system in peripheral arterial disease 2. Variations in factor VIII components (FVIII:C ; FVIIIR:Ag ; FVIII:WF) before and after venous occlusion

Factor VIII coagulant activity (FVIII:C), ristocetin co-factor or Willebrand Factor activity (FVIII:WF) and antigen related to FVIII (FVIIIR:Ag) were measured in a group of patients with peripheral arterial disease (PAD) and in a comparable control group. No differences were recorded between patients and controls both in basal conditions and after a standardized venous occlusion (VO) test. VO induced a significant increase in all the FVIII components, comparable in the two groups, with only minor variations in the ratios between them, due to a relatively greater response of the two activities with regard to the protein (FVIIIR:Ag) concentration. FVIII:C was significantly correlated with FVIIIR:Ag and with FVIII:WF only in controls in basal conditions, while no correlation was found in PAD group, nor after VO in either group.

Patients with atherosclerosis obliterans seem therefore to have normal levels of FVIII components and to mantain a normal capacity of responding to VO stimulation. FVIII coagulant activity can be acquired in local blood, or, in alternative, FVIII complex can be released as a whole from vascular walls following venous stasis.     

Combined aspirin and sulfinpyrazone in the prevention of recurrent hemodialysis vascular access thrombosis

We carried out a pilot study in 15 hemodialysis patients with recurrent vascular access thrombosis to examine whether the combination of low dose aspirin (85 mg once daily) and sulfinpyrazone (200 mg three times daily) is safe and effective in the prevention of vascular access thrombosis. Hemostatic measurements were performed prior to and after four weeks of starting the drug combination. Baseline values for fibrinopeptide A were elevated in all patients while those for platelet factor 4, fibrinogen, antithrombin III and protein C were generally within normal limits. A major reduction in the frequency of vascular access thrombosis from 0.114 per month to 0.04 per month was noted during combined drug treatment (p < 0.001). Although in vitro platelet aggregation to various stimuli was markedly suppressed and platelet thromboxane B₂ formation was almost completely inhibited in patients on aspirin/sulfinpyrazone, this was not associated with a significant further prolongation of the bleeding time. A relatively high rate of complications, particularly mild gastrointestinal bleeding, was noted in patients on aspirin/sulfinpyrazone that could not be predicted on the basis of pre-treatment hemostatic test results.     

The influence of age and smoking on hemostatic parameters in the chinese people

Plasma plasminogen activator inhibitor-1 (PAI) antigen, fibrinogen, factor VII, cholesterol, triglyceride (TG) and glucose were determined in 101 healthy subjects, who were divided into 10 subgroups according to age and smoking status. Factor VII and cholesterol were significantly higher in 50 healthy smokers than in the remaining 51 healthy non-smokers (p=0.037 and 0.008, respectively). By dividing smokers and non-smokers each into 5 different age groups, we found that smoking could significantly increase PAI and/or factor VII, but not fibrinogen, in several age groups. On the other hand, age would significantly increase fibrinogen in either smokers or non-smokers, and this increase would be promoted by smoking. In conclusion, smoking could produce higher factor VII or PAI, whereas age could effect the significant increase of fibrinogen which was further promoted by smoking. Though aging cannot be avoided, smoking should be abstained especially in the old people to prevent the occurrence of thromboembolic diseases.     

Platelets of patients with peripheral arterial disease are hypersensitive to heparin

We sought to verify earlier reports of increased platelet reactivity in patients with peripheral arterial disease (PAD) during perioperative heparin administration, and to test the hypothesis of platelet hypersensitivity to heparin in these patients. Before and after incubation of platelet rich plasma with unfractionated (UH), low molecular weight heparin (LMWH), and a low molecular weight heparinoid, real-time quantitative assessment of platelet function was performed by stagnation point flow adhesio-aggregometry (SPAA) in 21 patients with PAD and 14 healthy volunteers. With SPAA the occurrence of spontaneous aggregation is pathological. In the 15 patients requiring operation, platelet function and count were measured at regular intervals. To detect heparin dependent antibodies, the heparin induced platelet activation assay (HIPA) was performed preoperatively and after 10 days of heparin therapy. Mean baseline platelet adhesion in patients was double that observed in controls (p < 0.001). Spontaneous aggregation was seen in 9 (43%) patients and no controls (p < 0.001). In controls heparinoid reduced, whereas UH and LMWH slightly increased adhesion. Spontaneous aggregation was observed once with UH. Platelets from patients showed significantly enhanced adhesiveness and aggregability (p < 0.05) with UH and LMWH when compared to controls. Effects with the heparinoid were less pronounced and non-significant. In patients requiring operation, postoperative increases in platelet function and reductions in count were significant (p < 0.001). Ten (67%) experienced a fall in platelet count of > 50%. Preoperatively the HIPA assay showed no evidence of antibodies, whereas after heparin administration antibodies were verified in 4 (32%) patients and could not be ruled out in 6 (40%). Three developed postoperative thrombosis, in one case fatal. A hypersensitive in vitro and in vivo platelet response to heparin was verified in patients with PAD and a large number developed the immunological type of heparin-associated thrombocytopenia. Our findings suggest that a thrombin antagonist which does not interact with platelets may give the best perioperative protection in these patients.     

Clotting activation and impairment of fibrinolysis in malignancy

Different coagulation and fibrinolysis parameters were investigated in 149 patients with metastatic and non-metastatic tumours and results were compared with those obtained in a healthy population. Results showed a significant increase of thrombin-anti-thrombin complexes, fibrinopeptide A (FPA) and fibrin monomers in the group of patients (p< 0.001). There was also a significant prolongation of euglobulin lysis time (p< 0.005) and an increase of plasminogen activator inhibitor activity (p< 0.0001), fibrinogen degradation products (p< 0.001), and D-dimer (p< 0.05) in the group of patients as compared to controls; FPA levels were also increased in patients with metastases (p< 0.005). This study demonstrates clotting activation, at the level of fibrinogen to fibrin conversion, and impairment of fibrinolysis in patients with malignancy.     

Levels of plasma thrombomodulin are increased in atheromatous arterial disease

The plasma thrombomodulin (TM) level depends on the integrity of the endothelium and the clearance of the molecule. In several different pathological conditions, plasma TM levels increase with damage to the endothelium. We studied plasma TM levels in patients with various localizations of atheromatous arterial disease who had normal serum creatinine levels. Two groups of patients had a single symptomatic localization, which was either peripheral occlusive arterial disease (POAD) or ischemic heart disease (IHD) and a third group of patients had multiple symptomatic localizations (polyvascular). We compared the plasma TM levels with the plasma levels of other specific markers of endothelial cell activation such as: prostacyclin (PGI2), tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1). Plasma TM levels were significantly increased in all three individual groups and when all patients were considered (total patients), as compared with normal controls. When all patients were considered, there was a significant positive correlation between plasma TM levels and t-PA and between plasma TM levels and PGI2. A significant positive correlation was also found between the plasma TM levels and PAI-1 for patients with POAD. Thus, our findings suggest that an increased influx of TM into the plasma may be caused by endothelial cell damage in patients with atheromatous arterial disease. However in our study, the plasma TM levels obtained were similar for all three types of atheromatous arterial disease. Though plasma thrombomodulin is a marker of endothelial cell injury, it cannot be of a clinical interest until its levels are related to the extend of the atheromatous lesions. Moreover, we show that plasma TM is not independent of the usual endothelial cell activation markers.     

Blood coagulation and fibrinolysis in fertile women with previous thromboembolic complications and effects of venous occlusion

Blood coagulation and fibrinolysis were studied at rest in 35 fertile women with previous thromboembolic complications (TE) (group P) and in 20 healthy fertile women (group C). The effect of venous occlusion was studied in 23 and 15 individuals in each group. Before and after venous occlusion fibrinopeptide A (FPA) levels were higher and plasminogen activator (PLA) activity lower in group P. During venous occlusion factor VIII:C and fibrinogen increased in the stased blood in group P. FPA, factor VIIIR:Ag, antithrombin III (AT), PLA, ₂-antiplasmin and urokinase inhibitors increased in both groups. Decreased PLA activity was noted about twice as often among smokers as among non-smokers. No single variable suitable for screening was found but a combination of FPA, fibrinogen, factor VIII, AT and PLA maybe useful in diagnosing increased risk for TE. In some patients and a single control increased levels of inhibitors of the fibrinolytic system were found.     

Flow mediated fibrin thrombus formation in an endothelium-lined model of arterial branching

In vivo arterial thrombosis occurs preferentially at curvatures and branchings, i.e. regions of flow separation and recirculation where blood is retained orders of magnitude longer than within straight vessel sections. To examine the effect of such disturbed flow on endothelial thromboresistance glass T-branchings lined with endothelial cells from human umbilical cord veins (HUVEC) were perfused with buffered fibrinogen solution (3mg/ml). The flow was adjusted to form a large recirculation zone and flow conditions were determined beforehand by means of flow visualization via dye injection as well as by laser ultramicroscope anemometry. Thrombus formation, which was registered on-line by video and evaluated planimetrically, was induced by injection of thrombin at concentrations ranging from 0.3 to 2.0 units/ml. Fibrin thrombus growth always began within the flow niche at the point of flow separation and extended downstream along the wall and into the vessel lumen finally occluding up to 80% of the lumen. Light and electron microscopy revealed that overall thrombus form as well as the orientation of single fibrin fibers were correlated strictly to the prevailing streamlines. Despite the integrity of the endothelial lining fibrin thrombus formation occured. The fibrin fibers closely contacted the endothelial surface. These results indicate that recirculation zones promote fibrin thrombus formation sufficient to obstruct the vessel lumen and that intact endothelium alone is insufficient in preventing adhesion of fibrin to its surface.     

Determination of urokinase in the urine of healthy volunteers and patients with renal diseases

A sensitive enzyme immunoassay was developed for the determination of urokinase in the plasma and urine. Normal human urine contained 2068 ± 0.36 u/ml of UK. Gel filtration of the mixture of normal urine resulted in 2 peaks in the profile, one with molecular weight of 32, 000 and the other with molecular weight of 22, 000. Patients with glomerulonephritis had higher UK levels. Kidneys with minimal changes and mild type or with the inactive phase of systemic lupus erythematosus (SLE) secreted relatively high levels of UK in the urine.     

Antithemostatic and antthrombotic effects of capsaicin in comparison with aspirin and indomethacin

Capsaicin prolonged the tail bleeding time in the conscious mice. This effect was dose-dependent and maximal effect was observed at 3 hr after the oral administration. Capsaicin was effective in preventing death caused by ADP-induced acute pulmonary thromboembolism in mice at dose of 25 mg/kg, while aspirin and indomethacin had no effect at 200 mg/kg. Capsaicin also reduced the mortality in collagen- and sodium arachidonate-induced thromboembolic death at dose of 25 and 50 mg/kg, respectively, and aspirin and indomethacin were also effective in these models but only when the dose was higher than 200 mg/kg. Capsaicin, aspirin or indcmethacin could not protect mice from endotoxin shock. Capsaicin was found to suppress platelet aggregation markedly, but did not affect blood coagulation. In conclusion, capsaicin was proved to be more effective than aspirin and indomethacin in preventing the death of acute pulmonary thromboembolism, and this effect could be due to its inhibition on platelet aggregation.     

Evidence for the role of dialysis hypoxemia in the pathogenesis of hemodialysis-induced rise in tissue-type plasminogen activator

It is well known that hemodialysis (HD) causes a rise in plasma tissue-type plasminogen activator (t-PA). Although there have been several suggested mechanisms responsible for this effect of HD, the precise cause has not been well understood yet. Another complication of HD, when performed with acetate-containing dialysate, is hypoxemia, which is commonly observed during the first hour of the session. The purpose of this study was to investigate the relationship between dialysis hypoxemia and HD-induced t-PA changes during the first two hours of HD.

HD caused significant increase in plasma t-PA antigen levels. When individual t-PA profiles versus time were examined, two patterns were observed. Whilst ten subjects (%56) experienced minimal or no increase, t-PA antigen level of the remaining eight subjects began to rise at 30 minutes and continued at that level up to 90 minutes, when the last samples were drawn. The courses of pO₂ were also different; whilst the former group had “early-onset and short-term” hypoxemia, the latter had “late-onset and prolonged” hypoxemia. The amount of increase in t-PA antigen and the amount of decrease in pO₂ were correlated at 60 and 90 minutes of the HD session.

Thus, it is concluded that dialysis hypoxemia may contribute to HD-induced rise in plasma t-PA levels. Further studies comparing different dialysates and dialyser membranes are required to confirm this hypothesis.