TNF-a induces changes in endothelial cell functions, such as upregulation of tissue factor, resulting in endothelial procoagulant activity which may play a role in disseminated intravascular coagulation. The procoagulant activity of TNF-a-stimulated endothelial cell monolayers was studied in a human ex vivo native (nonanticoagulated) blood flow system using the three thrombin inhibitors recombinant hirudin, Ro 46-6240, and heparin. Under venous blood flow conditions (shear rate 65 s-') recombinant hirudin, Ro 46-6240, and heparin inhibited fibrin deposition on the endothelial cells by 50% at concentrations of 14, 28, and 412 ng/ml, respectively. The highest tested concentrations of the thrombin inhibitors reduced the postchamber fibrinopeptide A levels from 713±69 to < 70 ng/ml. Surprisingly, even at relatively high inhibitor concentrations, some local fibrin deposits were found on TNF-a-stimulated cells, suggesting that some endothelial cells possess higher procoagulant activity than others. Therefore, the surface expression pattern of tissue factor, the primary initiator ofcoagulation in this system, was examined by immunogold-silver staining. The results showed that the tissue factor density on the cell surface varied strongly among TNF-a-stimulated endothelial cells. Using TNF receptor-selective agonistic mutants ofTNFa, it was demonstrated further that the heterogenous surface expression of tissue factor was mediated entirely by the 55-kD TNF receptor and did not involve the 75-kD TNF receptor. We conclude that in this system TNF-a induces heterogenous tissue factor expression which may lead to a high local thrombin concentration, such that even in the presence ofthrombin inhibitors focal fibrin deposition occurs. (J. Clin. Invest. 1994.

Patients whose platelets are deficient in glycoprotein (GP) Ib, IIb- IIIa (thrombasthenia), or granule substances (storage pool deficiency, SPD) were studied to define further the properties of platelets that mediate platelet adhesion and thrombus formation on subendothelium. Both nonanticoagulated and citrated blood were exposed to everted, de- endothelialized rabbit vessel segments under controlled flow conditions and shear rates varying from 650 to 3,300 sec-1. Morphometry was used to measure platelet thrombus dimensions and the percentage of the subendothelial surface covered with contact (C) or spread (S) platelets. Adhesion was defined as C + S. The results in SPD demonstrated (1) reduced thrombus dimensions in delta-SPD (pure dense granule deficiency) in proportion to the magnitude of the dense granule defect; (2) an even greater reduction in thrombus dimensions in patients with combined deficiencies of alpha and dense granules (alpha delta-SPD); and (3) impaired platelet adhesion at several conditions in alpha delta-SPD and, in delta-SPD, a hematocrit-dependent impairment of adhesion in citrated blood at 2,600 sec-1. In thrombasthenia, platelets were present as a monolayer on the subendothelial surface in both nonanticoagulated and citrated blood, indicating an absolute requirement for GPIIb-IIIa in promoting platelet-platelet interaction at all shear rates and perfusion times. Two types of abnormalities in platelet-vessel wall interactions were observed. In nonanticoagulated blood, the percentage of platelets in the C phase was consistently increased at all shear rates, but C + S values were normal. These observations indicate that platelets deficient in GPIIb-IIIa do not spread normally on the subendothelial surface exposed to nonanticoagulated blood. With citrated blood, the C + S value in thrombasthenia was reduced at both 800 and 2,600 sec-1, as in von Willebrand's disease, and a similar degree of reduction (about 50%) was observed in normal blood treated with a monoclonal antibody to GPIIb- IIIa. The findings, together with theoretical considerations, are consistent with an hypothesis that GPIIb-IIIa mediates the spreading of platelets on subendothelium following the initial attachment through GPIb and that GPIIb-IIIa may be considered an adhesion site on the platelet membrane. Abnormalities of GPIIb-IIIa may, depending on the conditions of study, result in either increased values of C platelets or decreased values of C + S. The results of the study further suggest that a complex interaction of platelet granule factors and membrane GP mediate platelet adhesion and thrombus formation.

SUMMARY1. Blood platelets containing different amounts of 5-hydroxytryptamine (5-HT) were produced in vivo by the injection of5-hydroxytryptamine or of reserpine into normal rabbits and of 5-hydroxytryptamine into reserpinized rabbits. Before and after these injections the aggregation of platelets was measured in vitro.2. Platelets of untreated rabbits were aggregated by adenosine diphosphate (ADP) and by 5-hydroxytryptamine; (-)-adrenaline alonQ did not produce aggregation but markedly increased aggregation by 5-hydroxytryptamine.3. Platelets saturated with 5-hydroxytryptamine in vivo were no longer aggregated in vitro by 5-hydroxytryptamine or by 5-hydroxytryptamine plus adrenaline, but their aggregation by ADP was unchanged.4. Platelets from reserpinized rabbits lost about 99 % of their 5-hydroxytryptamine; the aggregation of these platelets did not differ significantly from that of platelets from control rabbits.5. Platelets from reserpinized rabbits injected with 5-hydroxytryptamine were aggregated neither by the amine alone nor by 5-hydroxytryptamine plus adrenaline, although these platelets contained much less 5-hydroxytryptamine than saturated platelets and only about one tenth as much as platelets from untreated rabbits.6. The findings support the hypothesis that the inhibitory effect of 5-hydroxytryptamine administered in vivo on platelet aggregation in vitro is due to the association of the amine with the platelet membrane.

Platelet glycoprotein (GP) IIb-IIIa inhibitors may become useful antithrombotic agents. Ro 4–5054 is a low molecular weight, noncyclic, peptidomimetic inhibitor that is three orders of magnitude more potent than RGDS in inhibiting fibrinogen binding to purified GPIIb-IIIa and in preventing platelet aggregation. Comparisons of RGDS and Ro 4–5054 in cell adhesion assays showed that, in contrast to RGDS, Ro 4–5054 was highly selective GPIIb-IIIa inhibitor. Effects of RGDV and Ro 4– 5054 on the conformation and activation state of GPIIb-IIIa were also examined. RGDV and Ro 4–5054 induced conformational changes in purified inactive GPIIb-IIIa as determined by binding of the monoclonal antibody D3GP3 (D3). These conformational alterations were not reversed after inhibitor removal, as indicated by the continued exposure of the D3 epitope and a newly acquired ability to bind fibrinogen. Similarly, RGDV and Ro 4–5054 induced conformational changes in GPIIb-IIIa on the intact platelet. However, after removal of the inhibitors, exposure of the D3 epitope was fully reversed and the platelets did not aggregate in the absence of agonist. Thus, while RGD(X) peptides and Ro 4–5054 transformed purified inactive GPIIb-IIIa into an irreversibly activated conformer, the effects of these inhibitors were reversible on the intact platelet. This suggests that factors present in the platelet membrane or cytoplasm may regulate in part the ability of the complex to shift between active and inactive conformers.

SUMMARY1. The aggregation of blood platelets was measured in vitro at different time intervals after the addition of 5-hydroxytryptamine (5-HT) or of reserpine to platelet-rich plasma of untreated rabbits and of rabbits injected with reserpine and 5-HT, i.e. while the platelets were taking up 5-HT or releasing it under the influence of reserpine.2. When 5-HT was added to stirred platelet-rich plasma the platelets aggregated reversibly within 1 min. The velocity of aggregation increased with 5-HT concentrations of 0*1-30 /tM and decreased with higher concentrations.3. (-)-Adrenaline, which alone did not produce aggregation, markedly accelerated the aggregation caused by 5-HT. The acceleration was greatest when 5-HT and adrenaline were added simultaneously.4. 5-HT added to the platelet-rich plasma in amounts that exceeded the 5-HT capacity of the platelets progressively diminished the velocity of aggregation produced by 5-HT plus adrenaline until aggregation was completely inhibited. Smaller amounts of 5-HT produced a transient inhibition of aggregation.5. The aggregation of platelets from reserpinized rabbits was inhibited by less 5-HT than the aggregation of platelets from normal rabbits.6. (-)-Adrenaline aggregated platelets of untreated rabbits but not those ofreserpinized rabbits or ofrabbits injected with 5-HT when reserpine was added in vitro 1-30 min previously.7. Platelets obtained from rabbits treated first with reserpine and subsequently injected with 5-HT were not aggregated by 5-HT plus adrenaline. During incubation in vitro these platelets progressively recovered their aggregability but this recovery was delayed by the monoamine oxidase inhibitor Pargyline.8. Imipramine in concentrations which did not influence platelet aggre-H. R. BAUMGARTNER gation by adenosine diphosphate (ADP) abolished aggregation produced by 5-HT or by 5-HT plus adrenaline.9. The inhibitory effect of adenosine on platelet aggregation was concentration-dependent and similar whether aggregation was produced by ADP, 5-HT or 5-HT plus adrenaline. 10. It is proposed that aggregation brought about by 5-HT is connected with its active uptake into the platelets and is caused by ADP formed from ATP during the active uptake of the amine. When 5-HT is no longer actively taken up, it also ceases to cause or to potentiate aggregation.