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After 35 years of research, a physiological regulator of platelet production has been identified and the recombinant protein is available. With the discovery of thrombopoietin (TPO), its potential use in a wide variety of clinical megakaryocytic and platelet disorders has been expected and clinical trials have been undertaken. To date, the reported encouraging pre-clinical studies indicate that, as with erythropoietin or G-CSF, minimal toxicity can be expected. A potential limiting side-effect of TPO could be the induction of thrombosis. Nevertheless, it is too early to know whether this cytokine will be of major therapeutic importance for patients with life-threatening thrombocytopenia, such as patients undergoing bone marrow transplantation or subjected to a high dose of chemotherapy. Several experimental and clinical studies are still needed to determine the efficacy of TPO in the prevention or the amelioration of bleeding, which is the ultimate goal for the appropriate use of cytokines with haemostatic benefit. Basic and clinical studies on regulators of megakaryocytopoiesis have rapidly progressed. Now, there is no doubt that some of these regulators are effective in correcting haematopoietic disorders of various aetiologies. Studies on negative regulators not only are important to understand the regulation of megakaryocytopoiesis in normal and pathological states but also have a potential clinical application. Some of these regulators have been shown to be effective in the treatment of essential thrombocythaemia and other myeloproliferative disorders. Platelet factor 4 (PF4) and some other chemokines are also capable of protecting progenitor cells from the cytotoxicity of chemotherapeutic drugs. However, detailed investigations are still required to determine the precise mechanism(s) of action of these regulators and to establish the optimal clinical protocols of negative regulators alone or in association with positive regulators for the treatment of various haematological diseases and cancer.

KRDS (Lys-Arg-Asp-Ser), a tetrapeptide from human lactotransferrin, was tested for its effects in vitro on dog platelet function and in vivo on femoral arterial thrombus formation in dogs. KRDS inhibited ADP (8 microM)-induced platelet aggregation (IC50: 350 microM) and arachidonic acid (2 mM)-induced thromboxane B2 generation (IC50: 175 microM). In addition, the thrombin (0.2 U/ml)-induced serotonin release was inhibited by KRDS (IC50: 525 microM) and the expression of alpha-granule membrane protein (GMP-140) was also inhibited (IC50: 350 microM). The results show that KRDS is an inhibitor for platelet aggregation and secretion to which the inhibition is more potent. Meanwhile, in the experiment of arterial thrombosis in dogs, KRDS (5 microM/kg) and 125I-SZ-51 (a monoclonal antibody against GMP-140) were injected before operation and immediately after the thrombus formation, respectively. In the KRDS group, the weight of removed thrombi was reduced to 50% of that in controls and the radioactivity per mg of labeled thrombi to 33.3% while in blood the radioactivity increased 2 times that in controls at the 4th hour after the injection of 125I-SZ-51. The radioactivity ratio between removed thrombi and blood was only 16% of that in controls. These results indicate that KRDS can inhibit thrombus formation in vivo and is a promising antithrombotic agent.

After an in vitro incubation of platelets with fibrillar collagen, their elastase activity is markedly and rapidly increased while proelastase decrease: proelastase is activated in situ into elastase which is released in its active form from the platelet. The activation of proelastase is likely due to the action of a trypsin-like enzyme present in the platelet. This protease has the same type of localization as proelastase and elastase: their highest activity is associated with light granules but part of these enzymes (or precursor) is also associated with the membranes. The mechanism of the arterial elastolysis induced by the platelets probably involves their adhesion to intimal thrombogenic surfaces (collagen) followed by a reaction during which proelastase would become available to the trypsin-like enzyme and would be activated into elastase directly released in the vessel wall.

We have measured the binding of 14C-ADP to isolated human platelet membranes by a technique using 0.8-mum Millipore filters to separate unbound tracer from membrane-bound nucleotide. The binding was dependent on the time of incubation and on the nucleotide concentration in the medium. The affinity constant was found to be comprised between 0.35 x 10(6) and 0.55 x 10 (6) M-1. Platelet membranes prepared from different thrombasthenic patients bound 14C-ADP with the same kinetic parameters as those from normal subjects. The affinity constant as determined for two of these thrombasthenic platelet membrane preparations was in the normal range.

High circulating levels of coagulation factor VII (FVII) are known to be associated with elevated concentrations of blood lipids. More specifically, hypertriglyceridemia is correlated with raised FVII coagulant activity (FVIIc). Recently, evidence has been described which suggests that elevation in FVIIc might reflect an increase in the total concentration of FVII, as evaluated by quantitation of FVII antigen (FVIIag). It is also known that FVIIc represents a risk factor for cardiovascular death, but the prediction of cardiovascular risk based on triglyceride estimation is the subject of conflicting results. Since dyslipidemia featuring abnormal triglyceride metabolism is pathophysiologically heterogeneous, we analyzed the possible relationships between FVII and triglyceride further and under standardized postprandial conditions. For this purpose we studied FVIIc and FVIIag in relation to triglyceridemia after a standardized test meal. Our results confirm that FVIIc and FVIIag levels are strongly correlated with each other (r = 0.714; p = 0.01). In addition, both were significantly increased (p less than 0.02) in patients who exhibited abnormal triglyceride levels 8 h after a standardized test meal, as compared to those who displayed normal triglyceridemia. Furthermore, we found that apolipoprotein B levels were also increased in such patients. The deficient postprandial catabolism of triglycerides, therefore, appears to be related to an increase in total FVII concentration, suggesting that some association between the metabolism of triglyceride-rich lipoproteins and FVII might underlie the mechanism of the elevation in FVII. Given the important contribution of FVII to hypercoagulability, then our results may be relevant to the understanding of the role of postprandial triglyceride in atherogenesis and in consideration of the circadian prevalence of cardiovascular thrombosis.

There is evidence that the increase in coagulation factor VII (FVII) represents a predictive risk factor of arterial thrombosis in coronary heart disease. Its relative contribution to this multifactorial process and its relationship to other risk factors, namely cholesterol and triglycerides, is yet a matter of investigation. In this study we aimed to clarify whether FVII synthesis or activation correlated with plasma lipid concentrations. For this, we assayed the plasma levels of FVII antigen (FVII:ag) and FVII coagulant activity (FVIIc) in types IIa, IIb and IV hyperlipidemic individuals, together with the levels of cholesterol, triglycerides, high-density lipoproteins and apolipoprotein B. FVII activation state (FVIIa) was then assessed by FVIIc/FVII:ag. In order to assess the possible correlation of FVII levels with the generation of thrombin and formation of fibrin, we also assayed the plasma concentration of fibrin degradation products (D-dimers) in these patients. Considering all the patients studied, there was a fair correlation between FVIIc and FVII:ag (r = 0.704; p less than 0.01). The mean levels of FVIIc and FVII:ag were significantly higher in type IV hyperlipidemia than in controls (t = 4.260; p less than 0.001 and t = 3.015; p less than 0.01, respectively) and other types of hyperlipidemia. We also found that FVIIc and FVII:ag significantly correlated to triglyceride concentration. We could not detect an evident activation of FVII in these patients since FVIIc/FVII:ag was not elevated in comparison with controls, nor did it correlate with any of the lipid determinations in any of the types of hyperlipidemia studied.(ABSTRACT TRUNCATED AT 250 WORDS)

A new technique, which simulates primary haemostasis in vitro was tested using blood from control persons and from patients with a defined abnormality of primary haemostasis. The device allows the reproducible measurement of in vitro bleeding time and volume in small samples of whole blood. Inhibition of platelet adhesion and aggregation can very sensitively be detected with the new technique, which allows its possible use in clinical or pharmacological applications.

Normal and thrombasthenic platelet membranes are able to specifically bind 14C-ADP (12). According to the concentration of ADP present in the medium, 'high affinity binding sites' (Ka = 0.5 X 10(6) M-1 and 'low affinity-binding sites' (Ka = 0.05 X 10(6) M-1) can be recognized. In the present study, dissociation of ADP from the 'high affinity binding sites' was measured with six normal and with three thrombasthenic platelet membrane preparations. A 1:100 dilution was used to dissociate the membrane-bound 14C-ADP and the kinetics of the dissociation was determined. The same profile of dissociation (with T 1/2 = 4--10 min at 37 degrees C) was observed using normal or thrombasthenic membranes. With both preparations, the rate of dissociation was increased (up to T 1/2 = 1--3 min) when unlabelled ADP (at concentration higher than 10(-5) M) was added in the diluting medium. The results confirm the presence of normal ADP binding sites on thrombasthenic platelet membrane and possibly suggest the existence of cooperative interactions between the sites on normal as well as on thrombasthenic membranes.

The binding of adenosine and adenylnucleotides to the human platelet membrane has been measured and the subsequent effect on the binding of 14C-ADP has been determined. The following conclusions have been drawn: (1) Two reactions with different velocities (t 1/2 = 15--30 sec and t 1/2 = 4--6 min) seem to be involved in the binding of ADP to the platelet membrane. (2) AMP binds to the platelet membrane and partially inhibits binding of ADP in a competitive manner. These two findings suggest that ADP binding sites on the platelet membrane may be of more than one type. (3) At concentrations up to 10(-5) M, ATP does not itself bind to the platelet membrane but is rapidly transformed into ADP which, in turn, binds to the membrane and inhibits the binding of 14C-ADP. (4) Adenosine does not significantly interfere with the binding of 14C-ADP and has not been shown to bind to the platelet membrane.