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Aims: To compare by a prospective study in high risk polycythemia vera (PV) patients 33P alone and 32P followed by low-dose hydroxyurea (HU) maintenance therapy. Toxicity, efficiency, and leukemogenic potential were studied., Patients: 483 patients with a documented PV, aged more than 65 years at diagnosis, were included between 1980 and 1996 in a prospective study comparing 32P alone and 32P followed by low-dose HU maintenance therapy. Blood cell counts were performed every two months and a clinical evaluation by a specialist was obtained every four or six months., Results: Treatments were well tolerated, but chronic leg ulcers were observed in the maintenance therapy arm. The risk of leukemia was about 15% at the 15th year in the group of patients treated by 32P alone, but reached 30% in the group receiving maintenance therapy. In both arms, there was no significant correlation between occurrence of leukemia and the total dose of 32P. There was a correlation between the leukemic risk and disease severity, estimated on the frequency of relapse. Cancer occurrence was slightly higher than expected in the maintenance arm. HU treatment did not protect against progression to myelofibrosis, probably due to the lack of maintenance of an efficient myeloid or megakaryocytic suppression. Median life-span was slightly shorter in the group receiving HU maintenance. In all cases, life-span was only one year lower than that observed in the reference population., Conclusion: For all these reasons, we suggest the us of 32P alone in elderly patients; complementary chemotherapy should only be prescribed in the cases with short-term relapse, and late resistance to 32P.

Background: In the last few years, the association of deep vein thrombosis with frequent biological risk factors and genetic polymorphisms has significantly modified the field of venous thrombosis. In this study, we measured plasma homocysteine levels and tested the C677T methylenetetrahydrofolate reductase (MTHFR) mutation., Patients and Methods: Plasma homocysteine levels and test for C677T MTHFR mutation were performed in 120 consecutive patients with objectively diagnosed deep vein thrombosis, and in 120 controls., Results: We found a strong association between hyperhomocysteinemia and thrombosis (odd ratio: 2.43 IC 95% [1.27-4.7]). Conversely the C677T MTHFR gene polymorphism is only associated with hyperhomocysteinemia but not associated with thrombosis., Conclusion: This is a preliminary study to the ongoing international multicentric study of SNFMI (Société nationale française de m0+edecine interne) concerning hyperhomocysteinemia and venous thrombosis.

Diabetic patients (3% of the French population) have a higher risk of coronary and peripheral vascular disease than non-diabetic subjects and develop long-term microvascular renal and retinal complications. Abnormalities of coagulation, haemostasis and fibrinolysis have been demonstrated in diabetics and contribute to these complications. The prescription of antithrombotics is therefore common in these patients. Platelet antiaggregants (aspirin and ticlopidine) are effective in primary and secondary prevention in reducing the overall number of vascular events (reduction of 17% in the last ATC meta-analysis) and of coronary and cerebrovascular complications in particular. Two studies have shown a preventive effect of antiaggregants on diabetic retinopathy in its initial stages. With regards to the value of the use of these agents, there are few complications which may be limited by appropriate measures. One problem lies in the choice of aspirin dosage, most studies having been performed with high doses ranging from 500 to 1,300 mg per 24 hours. It is therefore difficult to recommend doses less than 300 to 500 mg per 24 hours. The prescription of anticoagulants (heparin, vitamin-K antagonists) is not associated with more problems in diabetics than in non-diabetics. The same applies to the use of thrombolytics in the acute phase of myocardial infarction: the risk of haemorrhages, especially intraocular, is only theoretical, only one case (regressive) having been reported to date. In conclusion, diabetes is more a priviledged indication than a contra-indication to the use of antithrombotic, platelet inhibitor, anticoagulant and thrombolytic agents.

The therapeutic strategy in patients with Essential Thrombocythemia (ET) is a difficult balance between the prevention of bleeding and thrombotic complications and the risks of drug side effects and toxicity. Major bleeding is rare and seem to be related to higher platelet counts: therefore, a platelet count over 1500 x 10(9)/L is generally regarded as an indication for cytoreduction. Thrombotic complications include microvascular occlusive symptoms, which are reversible with low-dose aspirin, and large vessels thrombosis. The risk of major thrombosis is higher in ET patients aged more than 60 ys. and with previous occlusive event. In this high-risk group, the non-alkylating agent hydroxyurea (HU) significantly reduces the rate of vascular complications and has emerged as the treatment of choice. However, the long-term risk/benefit of HU remains disputed because its leukemogenic potential has not been ruled out. This holds also for other myelosuppressive agents, such as busulphan and pipobroman. Other drugs of particular interest for young patients include recombinant alpha-interferon (IFN) and Anagrelide. Both of them are effective in lowering platelet count, but their efficacy in reducing clinical complications remains to be demonstrated. However, both IFN and Anagrelide have shown to have frequent and clinically important side effects. Thus, further clinical studies are required to establish their role in the strategy of ET patient treatment.

We report a patient with congenital factor VII deficiency who developed severe arterial thrombosis. A 63-year-old-woman presented low factor VII clotting activity, amidolytic activity and antigen level < 4%. Activated factor VII plasmatic level was < 0.03 ng/ml compared to 4 ng/ml for the control value. She developed severe aorto-iliac thrombosis. 7 d before the thrombotic event, factor VII replacement therapy had been infused. Successful low molecular weight heparin therapy led to total disappearance of the aorto-iliac thrombus without bleeding complications. This suggests that factor VII infusion might have a thrombogenic effect in vivo and might be responsible for thrombosis.

In this study, the clinical history of two patients with the gray platelet syndrome, a rare congenital disorder associating thrombopathia and myelofibrosis is recalled. Complementary studies on platelets and megakaryocytes were performed, mainly with an immunocytochemical approach. In gray platelets, a general decrease of alpha-granule proteins, including PF4, beta tg and PDGF was observed. The decrease in platelet mitogenic activity (PDGF) was confirmed by biological and radio-immunological measurements. An abnormally high level of these compounds was also found in the plasma. In megakaryocytes cultured from the bone marrow of these patients, alpha-granule proteins were normally expressed in early maturation stages, whereas they were found to be absent in the mature megakaryocytes. An alpha-granule membrane glycoprotein, GMP 140 has been studied in resting and thrombin stimulated gray platelets and was found to be normally expressed at the surface of stimulated platelets. GMP140 was studied in resting platelets by immunoelectron microscopy and found to be present in vacuole probably corresponding to empty granules. This observation allows to conclude that alpha-granule membrane is formed in the gray platelet syndrome, but that there is a storage defect of alpha-granule soluble proteins, possibly due to an abnormal targetting of these proteins to the alpha-granule. Synthesis and subsequent release of these proteins, namely of the mitogenic factors, which can induce myelofibrosis and lung fibrosis by abnormal fibroblast stimulation, is discussed.

Studies have been carried out with cultured human megakaryocytes derived from Gray Platelet Syndrome blood and have demonstrated that MK-DGF and MK-F4 are present in the early stages of megakaryocyte maturation (day 5-6) but disappear in the late phase (day 9-11). In Gray platelets total mitogenic activity, PDGF and PF4 amounts were reduced while beta TG and PF4 plasma levels were slightly increased. These findings indicate a possible relationship between the loss of alpha granule contents from the megakaryocytes in the marrow and the myelofibrosis found in Gray platelet disorder and possibly associated in other diseases with platelet or megakaryocyte abnormalities.

The ultrastructure, cytochemistry, biochemistry, and functions of platelets from two patients with the familial gray platelet syndrome are described. Ultrastructural studies showed a lack of alpha-granules in the megakaryocytes in the vicinity of a marrow myelofibrosis and/or in platelets. A normal number of mitochondria and a slight increase of dense bodies was confirmed by labeling the whole patient with mepacrine. Platelet peroxidase (in the dense tubular system) and catalase-positive granules (revealed by the cytochemistry) were present. Platelets from both patients had severe deficiency of beta TG either after tritonization (less than 4% of normal) or thrombin treatment (less than 15% of normal), and the plasma beta TG levels were slightly increased. Functional studies of these platelets showed an uptake of [14C]5HT inhibited by reserpine similar to that in the control platelets. Thromboxane formation was within normal limits in the presence of arachidonic acid, ADP, collagen, or ionophore A23187, indicating that (1) the cyclooxygenase/thromboxane synthetase systems were not altered and (2) the phospholipase activities were not impaired. Although the platelet adhesion to prepolymerized fibrillar type III collagen and the ADP-, arachidonic acid-, and ionophore A23187-mediated aggregations were apparently normal, in every case the release of [14C]5HT was either at the low side of the normal range or decreased. This abnormality was increased when collagen or thrombin was added to either PRP or washed platelets from both patients, where at the same time, the aggregation and the release were markedly reduced. The results suggest that alpha-granules or their content has an influence on not only the platelet aggregation but also the dense bodies involved in the [14C]5HT release reaction.

Hemostasis disorders are involved in diabetic micro and macroangiopathy. Platelet hyperactivity is related at least in part to an imbalance between thromboxane A2 and prostacyclin. Both hyper and hypoglycemia result in platelet activation.

Fifteen patients (9 females and 6 males) with the "lupus type" circulating anticoagulant have been studied. The underlying disease was an auto-immune disorder in 11 cases and a malignant hemopathy in 4 cases. The manifestations frequently associated with the lupus inhibitor, such as thrombosis, thrombocytopenia and false-positive VDRL test were analysed. Hemorrhagic syndrome occurred only when thrombocytopenia or acquired abnormality of Willebrand's factor was present. Thrombotic events (8 cases) were frequent. Deep venous thrombosis was complicated with pulmonary embolism in 4 patients. Platelet abnormality, decreased fibrinolytic response or acquired Willebrand's syndrome were found in all patients with a thrombotic event. These different manifestations followed diverging courses in some patients with persistent thrombocytopenia although the anticoagulant had disappeared in 3 cases, negativation of the false-positive VDRL test while the anticoagulant remained unchanged in 1 case, occurrence of a thromboembolic episode although the anticoagulant had disappeared in 1 case.

To investigate the platelet contribution to the development of myelofibrosis in hairy cell leukaemia (HCL), we have studied two platelet alpha granule components in 15 patients with HCL before chemotherapy: mitogenic activity was measured by 3H thymidine incorporation in BALB/C 3T3 cells and beta thromboglobulin (beta TG) assayed by radioimmunoassay (RIA). Platelet mitogenic activity and beta TG content were significantly decreased in the patients as compared to the control subjects. The nine patients who were treated with recombinant human interferon (IFN alpha A) were restudied after 4 months of therapy. The levels of both mitogenic activity and beta TG platelet content were significantly increased after IFN alpha-treatment with a complete response in five of the nine treated patients, a partial response in two and no response in the two others. HCL seemed therefore to be responsible for an acquired platelet alpha granule defect. As in the grey platelet syndrome a relationship between this abnormal platelet granule storage and the development of myelofibrosis is suggested in HCL.

The authors report a case of chronic eosinophilic leukemia. Ristocetin did not aggregate patient's platelets in the absence of bleeding disorder. A possible relationship between arterial thrombosis observed in patients with eosinophilic leukemia and platelet abnormality is evoked.

In a child with acute megakaryoblastic leukemia--severe thrombocytopenia and myelofibrosis, EM studies on bone marrow showed a strict topographic relationship between the presence of clusters of abnormal megakaryocytes and the increased number of fibroblasts and extracellular fibers. Megakaryocytes and platelets lacked alpha-granules while the plasma thromboglobulin level was three times the normal level. This suggested that the alpha-granular proteins were synthesized but not retained in alpha-granules. If this occurs, the increased marrow levels of platelet-derived growth factor and factor 4 would favor the proliferation of fibroblasts and the synthesis of collagen, and thereby promote myelofibrosis. After therapy-induced remission, the number of marrow megakaryocytes decreased, the alpha-granules were normally produced, the plasma beta-thromboglobulin level was normal and the myelofibrosis disappeared. These observations suggest that during acute megakaryoblastic leukemia, an acquired gray-platelet syndrome occurs and that the local excretion of alpha-granule proteins triggers the myelofibrosis.

Platelets may promote the development of metastasis, and tumor cells that aggregate platelets are believed to be more malignant. We studied three different human mammary carcinoma cell lines, which had different interactions with human platelet-rich plasma (PRP). The MCF-7 and the T47-D cell lines induced an adenosine diphosphate (ADP)-mediated platelet aggregation. The third cell line, MDA-MB 231 did not induce any platelet aggregation. On the contrary, this cell line inhibited ADP- and arachidonic acid-induced platelet aggregation. This inhibiting activity is mainly adenosine-mediated. The mechanism by which platelets may contribute to the dissemination of cancer could be related to platelet growth factors. MCF-7 and T47-D cell lines induced a release of platelet-derived growth factor (PDGF). On the contrary, the MDA-MB 231 cell line did not induce any platelet release. The role of these platelet growth factors in tumor cell growth is discussed.

The authors have developed a sensitive immunoenzymatic method for assaying anti-cardiolipin antibodies in the serum of patients with lupus (SLE). These antibodies were present in the serum of 43/108 SLE patients, particularly in those patients with either false syphilis serology (p less than 0.02) or circulating anticoagulant (p less than 0.05). The mean titre of anti-cardiolipin antibodies was higher in the group with positive VDRL (less than 0.03). The anti-cardiolipin antibody titre was independent of the anti-native DNA antibody titre, but there was a correlation with the anti-denatured DNA antibody titre (p less than 0.02). This correlation can be partially explained by the antigenic similarity (phosphodiester bridge) between the two molecules. The preliminary clinical studies have not shown any correlation between the presence of anti-cardiolipin antibodies and the presence of signs such as thrombocytopenia, haemolysis, cerebral vascular accident, venous thrombosis, recurrent abortion. A longitudinal study of certain patients suggests that the anti-cardiolipin antibodies may disappear at the time of thrombotic accidents, which induces fixation of these antibodies to a platelet or vascular target and as a result of corticosteroid therapy.

Prelining graft material with autologous functioning endothelial cells might be one of the ultimate requirements to obtain a biocompatible surface. Accordingly, endothelial cells from stripped varicose veins were enzymatically harvested and grown on a fibronectin matrix. Proliferation was investigated in defined medium supplemented with various concentrations of endothelial cell growth supplement (ECGS) (25, up to 150 micrograms/ml) and heparin (10(-8), up to 10(-5)mol/L): optimal growth required both 150 micrograms/ml of ECGS and 10(-5)mol/L heparin. Under these conditions, cell culture achieved cell densities at a confluence of 1.2 +/- 1.1 10(5) cells/cm2 with a doubling time of 1 day. During subcultivation cultured cells consistently exhibited characteristic cobblestone morphology and immunofluorescent staining for factor VIII-related antigen, whereas prostacyclin production determined by enzyme-linked immunosorbent assay for 6-keto-prostaglandin F1 alpha reached 21.1 +/- 1.2 ng/10(6) cells after 15-minute stimulation with 1 U/ml of thrombin. Heparin-containing culture medium-endothelial cell interactions were particularly studied, and with iodine 125-heparin, binding was demonstrated with an apparent dissociation constant (Kd) of 0.36 +/- 0.04 mumol/L. A cold storage technique at -80 degrees C was sought, and freezed cells were used to coat in vitro polytetrafluoroethylene grafts. Protein-treated material allowed cell attachment and growth to a confluent monolayer as assayed by light and scanning electron microscopy. These data validate the feasibility of prelining grafts in vitro with autologous functioning endothelial cells. This approach may be useful in improving the performance of small-caliber vascular grafts according to prostacyclin production and surface-bound heparin of these cells.

The availability of fibrinogen receptors on platelets after ADP stimulation, was investigated in order to analyze platelet hyperaggregability induced by heparin. Unfractionated heparin increased the binding of fibrinogen on ADP-treated platelets. The results varied according to both the donor platelets and the kind of heparin preparation used. Beef lung heparin was more active than porcine intestinal mucosa heparin (P less than 0.001). Low molecular weight (LMW) heparin fractions however did not significantly increase the binding of fibrinogen to ADP-treated platelets. The effect of standard heparin and LMW heparin on the aggregation of normal platelets induced by the addition of plasma from patients with heparin-induced immune thrombocytopenia was also studied. Concerning heparin-induced immune thrombocytopenia we have shown that the plasma cofactor present in some patient plasma may induce platelet aggregation both in the presence of standard-heparin and in the presence of LMW heparin fractions. Therefore, one has to be careful when replacing standard heparin by LMW heparin or heparinoids since each patient may react differently.

Blood coagulation factors were measured in 9 patients under 55 years of age with retinal vein thrombosis. A statistical study of the results revealed 2 anomalies: reduced platelet aggregation in the presence of 1,2 M ADP and a decrease of plasminogen. It is difficult to correlate these biological anomalies with the clinical vascular accidents.

Venous thrombosis occurred frequently in Behçet's disease. In 12 patients with Behçet's disease of whom 6 have had leg venous thrombosis, some parameters of hemostasis or fibrinolysis have been studied. Platelet count, fibrinogen level, factor VIII coagulant activity, factor VIII antigen, plasminogen and antithrombin III level were in the normal range. Fibrinolytic capacity in response to venous occlusion of the arms was the same in patients and in healthy subjects, in the patients, there was no difference in regard to the occurrence of venous thrombosis. These parameters seemed therefore not able to detect the thrombotic tendency in this disease. These results are discussed with the other reports of the literature.

Platelet functions studied in 163 unselected diabetics compared with 163 controls had the following characteristics: hyperagregation induced by ADP (1.2 muM and 0.6 muM), delayed platelet disagregation (ADP: 0.6 muM), normal agregation in the presence of collagen and thrombin. Platelet hyperagregation induced by ADP was marked in both sexes in cases of retinopathy and in women after the age of 50. By contrast, no correlation was demonstrated between the degree of hyperagregation and age, weight, the duration of diabetes, blood glucose control, lipid profile, vascular complications other than retinopathy and the nature of treatment.

The diagnosis and treatment of apparently idiopathic recurrent thromboembolic disease (RTED) still raise difficult problems. However, recent data suggest that abnormalities of coagulation and fibrinolysis factors are important. Hereditary antithrombin III (AT III) deficiency or abnormal AT III, and hereditary protein C deficiency with autosomal dominant transmission have been associated with severe familiar RTED. More recently, we described a dysfibrinogenemia characterized by abnormal fibrin polymerization and abnormal plasminogen binding to the fibrin clot, responsible for familial RTED. Disorders of fibrinolytic activity in RTED are represented, in 70% of the cases, by reduced release or lack of production by endothelial cells of a vascular plasminogen activator. Hereditary plasminogen deficiency or abnormal plasminogen, although rare, are regularly responsible for RTED.

The present study was initiated to establish the functional factor V concentration in platelets of patients with a mild bleeding disorder ascribed to a gray platelet syndrome. This inherited platelet disorder has been characterized by a specific deficiency of alpha-granules and subsequent deficiencies in the alpha-granule proteins. We found that the concentration of plasma factor V was slightly decreased (70% of normal values). In contrast, platelet factor Va formation was severely impaired. Besides a much lower factor V content than in control platelets (10-20% of normal), the dependency of platelet factor Va formation on thrombin concentration was altered. Increasing the thrombin concentration 4-fold compared to the concentration that results in maximal factor Va generation from normal platelets did not result in a maximal factor Va formation from gray platelets. When a suspension of washed gray platelets was incubated with a prostacyclin analogue prior to the stimulation with thrombin, a 10-fold lower factor Va activity was measured. Thus, thrombin-induced factor Va formation in a suspension of gray platelets is the result of a release reaction, followed by the thrombin-catalyzed activation of released factor V. Whereas the kinetics of the former reaction are apparently impaired, the kinetics of the latter one were found to be identical to those observed for normal platelet and plasma factor V activation.