The Release of Tissue Factor Pathway Inhibitor and Platelet Factor 4 After Heparin Injection in Patients with Thrombocytosis.

Platelet factor 4 (PF4) and tissue factor pathway inhibitor (TFPI) are two proteins with high affinity for heparin. They are each stored in platelets, as well as on endothelial cell surfaces, from where both are displaced or released following an injection of heparin with a rapid and marked increase in serum levels. Prior work has demonstrated that the platelet count is one of the factors affecting the levels of heparin-releasable PF4. We therefore characterized the response to a dose of intravenous heparin previously demonstrated to completely displace PF4 from the non-platelet pool in subjects with normal or increased platelet counts. Seventeen patients with essential thrombocytosis (ET), 10 patients with polycythemia vera and high platelet counts (PV-H), 7 patients with polycythemia vera and normal platelet counts (PV-N) and 10 controls received an initial bolus of 40 I.U./kg of unfractionated heparin, followed 2 hours later by a 2nd bolus of a fixed dose of 1000 I.U. TFPI activity did not show any variation among the different groups, either before (TFPI) or after (HR-TFPI) the first bolus of heparin: ET, TFPI 92.6 ± 21.5%, HR-TFPI 298.3 ± 165.8; PV-H, TFPI 91.5 ± 32.0, HR-TFPI 210 ± 1.0; PV-N, TFPI 69.4 ± 24.0, HR-TFPI 203.0 ± 79.0; C, TFPI 109.5 ± 33.5, HR-TFPI 234.0 ± 60.4. TFPI activity returned to basal values prior to the 2nd injection of heparin, which again elicited a rise in TFPI, albeit smaller due to the lower level of heparin injected. In contrast to the lack of any difference between groups with respect to TFPI, the level of heparin-releasable PF4 (HR-PF4) was significantly higher in ET and PV-H patients compared to PV-N patients or controls. However when normalized for platelet count, both PV-H and PV-N had HR-PF4 levels after the 1st heparin injection that were significantly higher than observed in ET patients (PV-H 1.163 + 0.108, PV-N 1.411 + 0.019, ET 0.737 + 0.086 ng/10/3 platelets) supporting an increased platelet activation in PV. Thus, although platelets contain approximately 5-10% of the total amount of TFPI in plasma, they do not affect the major intravascular pool of TFPI mobilizable by heparin. However, since the concentration at the site of vessel wall injury is enhanced several-fold, TFPI could play a role in competing with PF4 to limit thrombus formation in patients with high platelet count.

Tissue factor pathway inhibitor (TFPI) activity in uremic patients during hemodialysis.

We studied tissue factor pathway inhibitor (TFPI) activity during hemodialysis in 10 uremic patients who were not receiving anticoagulant for at least 120 minutes. TFPI activity before dialysis was normal (patients 107 +/- 5.8%, controls 104 +/- 4.5%). During extracorporeal circuit it rose progressively with a statistically significant difference, reaching a plateau between 60 and 120 minutes. Since thrombin induces a marked redistribution and release of TFPI from stimulated endothelial cells and platelets contain about 10% of TFPI activity that is secreted following activation it is possible that thrombin-induced release of TFPI by endothelium and platelets could account for the increased TFPI we found during hemodialysis. To investigate this possibility we measured during dialysis beta-thromboglobulin (beta-TG), thrombin-antithrombin complex (TAT) and prothrombin fragment 1.2 (F 1.2). The increased levels of beta-TG, TAT and F1.2 we noted during extracorporeal circuit are in keeping with this concept. One hundred eighty minutes after initiation of dialysis, by which time all patients were receiving heparin there was a further increase in TFPI (to more than 200% of baseline), due to the presence of the glycosaminoglycan. This was due the previously reported displacement by heparin of the major intravascular pool of TFPI, from endothelial cell surfaces.

Demonstration that venous occlusion fails to release von Willebrand factor multimers.

The acute simultaneous release of tissue plasminogen activator (t-PA) and von Willebrand factor (vWF) from endothelial cells in response to a variety of agonists including thrombin, DDAVP, histamine and adrenalin has been described. In the present study we investigated the effect of venous occlusion on the circulating levels of t-PA and vWF, as well as the molecular organization of vWF in 20 normal subjects. After occlusion a significant increase in plasma t-PA levels was observed even after the values were corrected for haemoconcentration. Venous occlusion also enhanced plasma vWF values, but the increase was abolished when the correction for haemoconcentration was introduced. Following venous occlusion, no circulating abnormally large vWF multimers were detected in the subjects studied. These forms are normally not present in the circulation and are released from endothelial cells through the regulated vWF pathway; their absence therefore seems to demonstrate that this pathway is not activated after venous occlusion. Since occlusion does not enhance vWF synthesis, the increase in vWF observed in the subjects investigated may be fully attributed to haemoconcentration.

Reduced fibrinolytic potential one year after kidney transplantation. Relationship to long-term steroid treatment.

Thromboembolic complications constitute an important risk in renal transplant patients, in whom a hypercoagulable state is associated with immunosuppressive treatment, and the presence of hypercoagulability and hypofibrinolysis specifically with cyclosporine. Hypercorticism secondary to steroid treatment has been associated with a thrombophilic state and the presence of a reduced fibrinolytic potential in particular. The aims of this study were to first evaluate the fibrinolytic potential by the venous occlusion (VO) test in 19 renal transplant (RT) patients, and then compare these findings with those obtained in similar groups of normal subjects and patients with Cushing's disease. The following tests were carried out before and after the VO test: euglobulin lysis time and t-PA and PAI-1 activities and antigen. Compared with normal controls, RT and Cushing's patients both showed a similar significant increase in PAI-1 activity and concentration. The VO test revealed a similar impairment in fibrinolytic potential in both the RT and Cushing groups. High and pathological PAI-1 levels before and after the VO test were consistent with a defective fibrinolytic potential due to the inhibitory effect of PAI-1 on plasminogen activation. A hypofibrinolytic state was found in 68.4% of RT patients. Our results suggest that an imbalance in the fibrinolytic system is a typical feature of RT patients one year after transplantation. Steroids appear to be the immunosuppressive drug mainly involved in determining thromboembolic risk after renal transplantation.

Fibrinolytic pattern in recurrent spontaneous abortions: no relationship between hypofibrinolysis and anti-phospholipid antibodies.

Patients with antiphospholipid antibodies may suffer from thrombotic events and recurrent spontaneous abortions. A defective fibrinolytic potential has been described in women with recurrent fetal losses. We investigated the prevalence of anticardiolipin antibodies and of various fibrinolytic abnormalities in 64 females with a history of at least two abortions of unknown origin. Anticardiolipin antibodies were present in the serum of 31 patients (48.4%). The overall prevalence of fibrinolytic disorders was 67.2% (43 cases) and resulted significantly higher than that of aCL positivity (P = 0.03). In most of cases the impaired fibrinolytic potential after venous occlusion test was due to increased PAI-1 levels; only in a few instances a defective fibrinolytic response was due to reduced t-PA release, a combined defect or an intrinsic fibrinolytic deficiency. After division of patients in two groups on the basis of the aCL presence, the distribution of different fibrinolytic defects was similar in aCL positive and negative women, suggesting the lack of correlation between hypofibrinolysis and aCL antibodies. Plasminogen abnormalities resulted compatible with congenital hypoplasminogenemia in two aCL negative women, whereas in four aCL positive patients they were suggestive for acquired dysplasminogenemia. Our results indicate that patients with recurrent spontaneous abortions may present fibrinolytic disorders, which occur independently and more often than aCL positivity. An accurate investigation of the fibrinolytic potential, and, namely, of PAI-1 levels, should be included in the study of females suffering from repeated fetal losses.

Protein C, factor VII and prothrombin time as early markers of liver function recovery or failure after liver transplantation.

Irreversible initial non-function of the graft liver is a life-threatening early complication of orthotopic liver transplantation (OLT), which needs immediate retransplantation if the patient is to survive. Since protein C (PC) is a vitamin K dependent protein synthesized in the liver and with the same half-life as factor VII (FVII), the behaviour of PC in patients undergoing OLT was studied in comparison with prothrombin time (PT) and FVII. Twelve OLT patients were divided into two groups on the basis of clinical outcome: group A (six cases) in which OLT was successful, and group B (six cases) who developed initial non-function of the graft liver. PT, FVII activity (FVII:act) and antigen (FVII:Ag) and PC activity (PC:act) and antigen (PC:Ag) were carried out on six blood samples collected during the operation. At baseline, coagulation disorders were in agreement with the underlying liver disease, but no differences were seen between the two groups when all tests were considered. Ten minutes, 1, 2 and 3 h after liver reperfusion, mean PT and FVII:act were always significantly increased in good responder patients compared to non-responders. FVII:Ag and PC:Ag were significantly higher in group A than in group B starting 2 h after the liver graft reperfusion; no difference was seen in PC:act levels between the two groups. In addition, PC:Ag mean levels were increased with respect to corresponding PC:act values in non-responder patients, suggesting a qualitative rather than quantitative defect of protein synthesis due to liver damage. In conclusion, PT and FVII:act were more sensitive than PC activity as early prognostic indices of clinical outcome in OLT.

Unusual thrombotic-like retinopathy (Coats' disease) associated with congenital plasminogen deficiency type I.

A new kindred with heterozygous plasminogen deficiency type I is described. The proband, a 17-year-old male, showed a peculiar thrombotic-like retinal picture compatible with Coats' disease. Extensive coagulation studies revealed decreased levels of both plasminogen activity and antigen to about 50% of normal values. Five out of 13 family members from the paternal side showed the same fibrinolytic defect. In two cases, a history of recurrent phlebites of the lower limbs was present. One unaffected patient also had a superficial phlebites at a young age; her plasminogen levels were shown to be within normal limits, but a long-standing oestroprogestinic intake could have influenced and normalized the results. No other family member showed retinal abnormality. This is the first case of hypoplasminogenaemia associated with Coats' disease. A possible role of the fibrinolytic defect in the pathogenesis of this unusual retinopathy is suggested. Finally, the occurrence of thrombotic manifestations in other affected family members supports the opinion that plasminogen deficiency should be considered as a potential risk factor for thrombosis.

Effect of recombinant-tissue plasminogen activator, low molecular weight urokinase and unfractionated heparin on platelet aggregation.

We studied the interaction of two thrombolytic agents, recombinant-tissue plasminogen activator (r-TPA) and low molecular weight urokinase (UK), with platelet aggregation in the absence or presence of unfractionated heparin (UH). With platelet rich plasma (PRP), a dose dependent inhibition was seen for both r-TPA and UK. However, this effect was more evident in the presence of r-TPA. UH did not modify this effect. However, it enhanced platelet aggregation induced by ADP and decreased the aggregation induced by collagen (COL) as already shown by us. With washed platelets, only r-TPA decreased platelet aggregation in a dose dependent manner in the presence of COL and only at the highest dose (100 micrograms) in the presence of TH. The presence of ten units of plasminogen (PLG) together with 10 micrograms of r-TPA or 1250 units of UK totally inhibited TH-induced platelet aggregation. UH reversed this effect. In contrast, when COL was the aggregating agent, the inhibition of platelet aggregation in presence of PLG, seems to be further increased by UH. Since UH is an adjunct of thrombolytic therapy to prevent rethrombosis, this double edged sword could partially explain the lack of therapeutic effect in some patients.

Interaction between histidine-rich glycoprotein and platelet factor 4 with dermatan sulfate and low-molecular-weight dermatan sulfate.

There is a controversy about whether or not histidine-rich glycoprotein (HRG), the most abundant plasma protein with glycosaminoglycans-neutralizing capacity, is able to prevent the inhibition of human thrombin by heparin cofactor II (HC II) in the presence of dermatan sulfate (DS). The authors studied the interaction of DS and low molecular weight DS, in a purified system with HRG, platelet factor 4 (PF 4), and with HC II. Their results show that HRG, like PF 4, has an affinity, not only for heparin, but also for DS. However, this affinity seems very weak. In fact, HRG is 10 times less effective than PF 4 in neutralizing the 50% antithrombin activity of HC II in the presence of DS.

Venous thrombosis and tissue plasminogen activator release deficiency: a family study.

Thrombotic events are often due to fibrinolytic defects such as impaired tissue plasminogen activator (tPA) synthesis and/or release or increased plasminogen activator inhibitor (PAI) levels. In this report we describe four members of a family with a history of recurrent venous thrombosis, who demonstrated defective tPA release after dynamic tests. Two symptomatic patients and one asymptomatic individual showed absent or abnormally low tPA antigen (tPA:Ag) and activity (PA) increases after DDAVP infusion and/or 20 min of venous occlusion. In these patients PAI values were slightly higher than controls. A satisfactory tPA:Ag release was found in the fourth asymptomatic patient. All other coagulation tests were within the normal ranges. This familial defect of the fibrinolytic system seems to be inherited as an autosomal trait.

Fibrinolytic effects of defibrotide in atherosclerotic patients.

Defibrotide is a polydeoxyribonucleotide salt that shows antithrombotic activity through a suggested profibrinolytic mechanism. To study the effectiveness of defibrotide in atherosclerosis, we evaluated the fibrinolytic and coagulation behavior in normal subjects and patients with atherosclerotic disease, before and after single or repeated intravenous defibrotide infusion. A significant shortening of the ELT was found in all subjects. However, since neither t-PA increase nor PAI decrease was observed, we suggest that the profibrinolytic response to defibrotide may be due to mechanisms other than t-PA stimulation. Our results provide further evidence for the usefulness of defibrotide antithrombotic prophylaxis in atherosclerosis.

Effect of buflomedil, dipyridamole and indobufene on hemorrheologic and blood coagulation parameters in patients who underwent vascular surgery for severe atherosclerosis.

We studied 30 patients (age range 58 to 64 years) who underwent vascular surgery for peripheral vascular disease (24 patients) and for carotid stenosis (6 patients). At the time of the study, all subjects have been taking antiplatelet agents for at least three months. They were divided into three groups of ten patients each: the first group received buflomedil (BUF), the second dipyridamole (DIP) and the third indobufene (IND), as treatment before vascular surgery. We investigated the influence of these drugs on hemorrheologic (hematocrit, whole blood and plasma viscosity) and blood coagulation (partial thromboplastin time, prothrombin time and bleeding time) parameters in all patients. In our experience, none of the three drugs influenced any tested parameters, which were within normal limits: among them we noted only a statistically significant difference in bleeding time between patients treated with BUF and IND.

Fibrinolytic effect of a particular glycosaminoglycan (endoglycan) per os on normals and in patients with chronic artery disease.

Endoglycan, a heparan-dermatan sulphate association, is a highly purified heparinoid extracted from porcine intestinal mucosa. The aim of our study was to investigate the fibrinolytic system in a group of healthy controls and vascular disease patients, before and after endoglycan administration "per os". All the patients had a reduced basal fibrinolytic activity. The tests carried out were PT, PTT, FDP, Euglobulin Lysis Time (ELT), fibrinogen, plasminogen, alpha 2-antiplasmin, alpha 2-macroglobulin and t-PA activity assayed with a chromogenic method. After endoglycan administration, we have shown a significant shortening of ELT with complete normalization during the treatment. A fibrinogen decrease and either plasminogen or alpha 2-antiplasmin increase was seen. This was shown in normals too, however to a lesser extent. During therapy most of the healthy subjects, but only some patients, showed increased t-PA levels. Before and during treatment, significantly higher t-PA levels were seen in the control group as compared to the patients group. Reduced t-PA release was seen in our vascular disease patients. In conclusion, endoglycan "per os" appears to exert a stimulatory effect on the fibrinolytic system.

[Erythrocyte filterability and relative viscosity in liver cirrhosis and chronic hepatitis]. / Filtrazione eritrocitaria e viscosità relativa nella cirrosi epatica e nella epatite cronica.

Fifty patients with liver disease, of whom twenty-four had chronic active hepatitis and twenty-three had liver cirrhosis, were studied and compared with thirty-nine age- and sex-matched controls. Whole blood, plasma and their relative viscosities and the main factors capable of influencing these parameters (Ht, fibrinogen, triglycerides, cholesterol, plasma glucose, total proteins and gamma-globulins) were tested. Whole blood filterability time and velocity of red blood cells (VRBC) were also determined. In ten patients deformability of erythrocytes was determined both in whole blood and in suspensions of RBC in buffer at a Ht of 10%. Whole blood filterability time was significantly increased in all subjects with liver disease. Corrected filterability time was increased only in the cirrhotic group associated with a significant decrease of Ht and fibrinogen and a slight increase in gamma globulins and plasma viscosity. Whole blood viscosity was in the normal range while relative viscosity was decreased in all patients. These findings suggest that both shape alterations of red cells (such as echinocytic shape) and modifications of plasma proteins probably lead to a decrease of red cell filtration in cirrhotic patients.

A DIC-like picture on plasma and ascitic fluid of cirrhotic patients.

Ascitic fluid reinfusion in severe cirrhosis has frequently been associated with intravascular coagulation (DIC). A low-grade DIC has been postulated to be present in liver cirrhosis. PT, APTT, fibrinogen, plasminogen, antiplasmin, fibrin degradation producers (FDP), euglobulin lysis time, tissue plasminogen activator, and fibrinopeptide A were investigated both in the plasma and ascitic fluid of cirrhotic patients before and after the concentration-reinfusion technique. Our results indicate that increased thrombin formation associated with hyperfibrinolysis is present in the plasma of cirrhotic patients. In ascitic fluid very high levels of thrombin and fibrinolysis activation were found. We conclude that (1) a DIC-like picture exists in ascites and (2) after ascites reinfusion procedures, ascitic fluid is the principal factor in the pathogenesis of DIC. During ascitic fluid reinfusion heparin treatment could be used successfully.

Excess of factor X antigen versus clotting counterpart in coumarin treated patients as determined by an Elisa method.

Factor X antigen was assayed by means of an Elisa (sandwich) method in 36 patients on long-term anticoagulant therapy. The average value observed was 31.4% +/- 12.2. In almost every instance the antigen level was higher than the clotting counterpart (14.4% +/- 4.5). In a few instances no major difference was noted between factor X antigen and factor X activity. The method correlated fairly well with other immunological methods (electroimmunoassay and Laser Nephelometer). Therefore, factor X Elisa method appears to be a suitable method for factor X antigen evaluation.

Antithrombin III deficiency: a report of 14 cases belonging to three different kindreds.

A hereditary deficiency of AT III is described in 14 subjects belonging to three different kindreds. There is no consanguineity in any of the families investigated. The pattern of inheritance of defect appears autosomal dominant. Seven of the affected subjects presented thrombotic episodes (deep vein thrombosis, splanchnic thrombosis, pulmonary embolization). The main laboratory features were: normal routine clotting tests, decreased AT III activity in all assay systems and concomitantly reduced AT III antigen levels. Crossed immunoelectrophoresis showed only reduced peaks with respect to normal in both plasma and serum. No correlation was found between age of patients and AT III levels.

The contact phase of blood coagulation and renin activation in essential hypertension before and after captopril.

The contact phase of blood coagulation in a group of patients suffering from essential hypertension was studied before and after captopril administration. The baseline levels of factor XII, factor XI and plasminogen were significantly higher than in normals and correlated with baseline diastolic blood pressure levels. On the contrary, plasma prekallikrein was not significantly different from normal. These results suggest the presence of a hypercoagulable state in essential hypertension. After captopril administration, factor XII, factor XI and prekallikrein rapidly decreased, perhaps as a consequence of the drug's effect on the vascular endothelial surface. There was no correlation between the changes of active and inactive renin and the changes of prekallikrein and plasminogen levels. Our data do not support the view that factor XII-plasma kallikrein or plasmin dependent pathways are involved in the activation of inactive renin in vivo. Captopril, by provoking rapid pressure changes, appears to be able to affect the clotting system.