Aaron J. Marcus

KAPOSI'S SARCOMA IN HOMOSEXUAL MEN—A REPORT OF EIGHT CASES

The clinical findings in eight young homosexual men in New York with Kaposis sarcoma showed some unusual features. Unlike the form usually seen in North America and Europe, it affected younger men (4th decade rather than 7th decade); the skin lesions wee generalised rather than being predominantly in the lower limbs, and the disease was more aggressive (survival of less than 20 months rather 8-13 years). All eight had had a variety of sexually transmitted diseases. All those tested for cytomegalovirus antibodies and hepatitis B surface antigen of anti-hepatitis B antibody gave positive results. This unusual occurrence of Kaposis sarcoma in a population much exposed to sexually transmissible diseases suggests that such exposure may play a role in its pathogenesis.

The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39.

We previously demonstrated that when platelets are in motion and in proximity to endothelial cells, they become unresponsive to agonists (Marcus, A.J., L.B. Safier, K.A. Hajjar, H.L. Ullman, N. Islam, M.J. Broekman, and A.M. Eiroa. 1991. J. Clin. Invest. 88:1690-1696). This inhibition is due to an ecto-ADPase on the surface of endothelial cells which metabolizes ADP released from activated platelets, resulting in blockade of the aggregation response. Human umbilical vein endothelial cells (HUVEC) ADPase was biochemically classified as an E-type ATP-diphosphohydrolase. The endothelial ecto-ADPase is herein identified as CD39, a molecule originally characterized as a lymphoid surface antigen. All HUVEC ecto-ADPase activity was immunoprecipitated by monoclonal antibodies to CD39. Surface localization of HUVEC CD39 was established by confocal microscopy and flow cytometric analyses. Transfection of COS cells with human CD39 resulted in both ecto-ADPase activity as well as surface expression of CD39. PCR analyses of cDNA obtained from HUVEC mRNA and recombinant human CD39 revealed products of the same size, and of identical sequence. Northern blot analyses demonstrated that HUVEC express the same sized transcripts for CD39 as MP-1 cells (from which CD39 was originally cloned). We established the role of CD39 as a prime endothelial thromboregulator by demonstrating that CD39-transfected COS cells acquired the ability to inhibit ADP-induced aggregation in platelet-rich plasma. The identification of HUVEC ADPase/CD39 as a constitutively expressed potent inhibitor of platelet reactivity offers new prospects for antithrombotic therapeusis.

Synthesis of Prostacyclin from Platelet-derived Endoperoxides by Cultured Human Endothelial Cells

We have previously shown that aspirin-treated endothelial cells synthesize prostacyclin (PGI(2)) from the purified prostaglandin endoperoxide PGH(2) (1978. J. Biol. Chem.253: 7138). To ascertain whether aspirin-treated endothelial cells produce PGI(2) from endoperoxides released by stimulated platelets, [(3)H]arachidonic acid-prelabeled platelets were reacted in aggregometer cuvettes with the calcium ionophore A 23187, thrombin, or collagen in the presence of aspirin-treated endothelial cell suspensions. This procedure permitted thin-layer radiochromatographic quantitation of [(3)H]PGI(2) as [(3)H]6-keto-PGF(1alpha) and [(3)H]thromboxane A(2) (TXA(2)) as [(3)H]TXB(2), as well as analysis of platelet aggregation responses in the same sample. In the presence of aspirin-treated endothelial cells, platelet aggregation in response to all three agents was inhibited. [(3)H]6-keto-PGF(1alpha) was recovered from the supernates of the combined cell suspensions after stimulation by all three agents. The order of PGI(2) production initiated by the stimuli was ionophore > thrombin > collagen. The amounts of platelet [(3)H]TXB(2) recovered were markedly reduced by the addition of aspirin-treated endothelial cells. In separate experiments, 6-keto-PGF(1alpha) and TXB(2) were quantitated by radioimmunoassay; the results paralleled those obtained with the use of radiolabeling. The quantity of 6-keto-PGF(1alpha) measured by radioimmunoassay represented amounts of PGI(2) sufficient to inhibit platelet aggregation. These results were obtained when 200,000 platelets/mul were combined with 3,000-6,000 aspirin-treated endothelial cells/mul. At higher platelet levels the proportion of 6-keto-PGF(1alpha) to TXB(2) decreased and platelet aggregation occurred. Control studies indicated that aspirin-treated endothelial cells could not synthesize PGI(2) from exogenous radioactive or endogenous arachidonate when stimulated with thrombin. Therefore the endothelial cell suspensions could only have used endoperoxides from stimulated platelets.Thus, under our experimental conditions, production by endothelial cells of PGI(2) from endoperoxides derived from activated platelets could be demonstrated by two independent methods. These experimental conditions included: (a) enhanced platelet-endothelial cell proximity, as attainable in stirred cell suspensions; (b) use of increased endothelial cell/platelet ratios; and (c) utilization of arachidonate of high specific activity in radiolabeling experiments. Furthermore, when a mixture of platelets and endothelial cells that were not treated with aspirin was stimulated with thrombin, more than twice as much 6-keto-PGF(1alpha) was formed than when endothelial cells were stimulated alone. These results indicate that endothelial cells can utilize platelet endoperoxides for PGI(2) formation to a significant extent.

Phospholipid Metabolism in Stimulated Human Platelets: CHANGES IN PHOSPHATIDYLINOSITOL, PHOSPHATIDIC ACID, AND LYSOPHOSPHOLIPIDS

Endogenous phospholipid metabolism in stimulated human platelets was studied by phosphorus assay of major and minor components following separation by two-dimensional thin-layer chromatography. This procedure obviated the use of radioactive labels. Extensive changes were found in quantities of phosphatidylinositol (PI) and phosphatidic acid (PA) as a consequence of thrombin or collagen stimulation. Thrombin addition was followed by rapid alterations in the amount of endogenous PI and PA. The decrease in PI was not precisely reciprocated by an increase in PA when thrombin was the stimulus. This apparent discrepancy could be explained by removal of a transient intermediate in PI metabolism, such as diglyceride, formed by PI-specific phospholipase C (Rittenhouse-Simmons, S., J. Clin. Invest.63: 580-587, 1979). Diglyceride would be unavailable for PA formation by diglyceride kinase, if hydrolyzed by diglyceride lipase (Bell, R. L., D. A. Kennerly, N. Stanford, and P. W. Majerus. Proc. Natl. Acad. Sci. U. S. A.76: 3238-3241, 1979) to yield arachidonate for prostaglandin endoperoxide formation. Thrombin-treated platelets also accumulated lysophospho-glycerides. Specifically, lysophosphatidyl ethanolamines accumulated within 15s following thrombin addition. Fatty acid and aldehyde analysis indicated phospholipase A(2) activity, with an apparent preference for diacyl ethanolamine phosphoglycerides. In the case of collagen, these changes occurred concomitantly with aggregation and consumption of oxygen for prostaglandin endoperoxide formation.THESE STUDIES OF ENDOGENOUS PHOSPHOLIPID METABOLISM PROVIDE INFORMATION SUPPORTING THE EXISTENCE OF TWO PREVIOUSLY POSTULATED PATHWAYS FOR LIBERATION OF ARACHIDONIC ACID FROM PLATELET PHOSPHOLIPIDS: (a) the combined action of PI-specific phospholipase C plus diglyceride lipase yielding arachidonate derived from PI; and (b) a phospholipase A(2) acting primarily on diacyl ethanolamine phosphoglyceride.

Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vitro.

Abstract Interactions of human platelets with neutrophils were studied in suspensions of [ 3 H]arachidonate-labeled platelets and unlabeled neutrophils stimulated with ionophore A23187. Several radioactive arachidonate metabolites, not produced by platelets alone, were detected, including [ 3 H]-labeled leukotriene B 4 (LTB 4 ), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When [ 3 H]12-HETE, a platelet product, was added to stimulated neutrophils, DHETE was formed. Similarly, when [ 3 H]5-HETE, a neutrophil product, was added to stimulated platelets, DHETE was the major product. These results suggest that upon stimulation: 1) platelet-derived arachidonate may serve as precursor for the neutrophil-derived eicosanoids LTB 4 and 5-HETE, and 2) that platelet-derived 12-HETE can be converted to DHETE by human neutrophils. The present investigation documents cell-cell interactions via the lipoxygenase pathway, which may be important in hemostasis, thrombosis and inflammation.

Enhancement of platelet reactivity and modulation of eicosanoid production by intact erythrocytes. A new approach to platelet activation and recruitment.

Erythrocytes are known to influence hemostasis. Bleeding times are prolonged in anemia and corrected by normalizing the hematocrit. We now demonstrate that intact erythrocytes modulate biochemical and functional responsiveness of activated platelets. A two-stage procedure, permitting studies of cell-cell interactions and independently evaluating platelet activation and recruitment within 1 min of stimulation, was developed. Erythrocytes increased platelet serotonin release despite aspirin treatment, enzymatic adenosine diphosphate removal, protease inhibition, or combinations thereof. The data suggested that erythrocyte enhancement of platelet reactivity can reduce the therapeutic effectiveness of aspirin. Erythrocytes metabolically modified platelet arachidonate or eicosapentaenoate release and eicosanoid formation. They promoted significant increases in cyclooxygenase and lipoxygenase metabolites upon platelet stimulation with collagen or thrombin. However, with ionophore, erythrocytes strongly reduced platelet lipoxygenation. These erythrocyte modulatory effects were stimulus-specific. Activated platelet-erythrocyte mixtures, with or without aspirin, promoted 3-10-fold increases in extracellular free fatty acid, which would be available for transcellular metabolism. Erythrocyte-induced increases in free eicosapentaenoate may contribute to antithrombotic and anti-inflammatory effects of this fish oil derivative. These results provide biochemical insight into erythrocyte contributions to thrombosis and hemostasis, and support the concept of thrombus formation as a multicellular event.

Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39.

Excessive platelet accumulation and recruitment, leading to vessel occlusion at sites of vascular injury, present major therapeutic challenges in cardiovascular medicine. Endothelial cell CD39, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ATP and ADP released from activated platelets, thereby abolishing recruitment. Therefore, a soluble form of CD39, retaining nucleotidase activities, would constitute a novel antithrombotic agent. We designed a recombinant, soluble form of human CD39, and isolated it from conditioned media from transiently transfected COS-1 cells and from stably transfected Chinese hamster ovary (CHO) cells. Conditioned medium from CHO cells grown under serum-free conditions was subjected to anti-CD39 immunoaffinity column chromatography, yielding a single approximately 66-kD protein with ATPase and ADPase activities. Purified soluble CD39 blocked ADP-induced platelet aggregation in vitro, and inhibited collagen-induced platelet reactivity. Kinetic analyses indicated that, while soluble CD39 had a Km for ADP of 5.9 microM and for ATP of 2.1 microM, the specificity constant kcat/Km was the same for both substrates. Intravenously administered soluble CD39 remained active in mice for an extended period of time, with an elimination phase half-life of almost 2 d. The data indicate that soluble CD39 is a potential therapeutic agent for inhibition of platelet-mediated thrombotic diatheses.

Erythrocyte Promotion of Platelet Reactivity Decreases the Effectiveness of Aspirin as an Antithrombotic Therapeutic Modality The Effect of Low-Dose Aspirin Is Less Than Optimal in Patients With Vascular Disease Due to Prothrombotic Effects of Erythrocytes on Platelet Reactivity

BACKGROUND Aspirin (acetylsalicylic acid, ASA) is widely used for secondary prevention of ischemic vascular events, although its protection only occurs in 25% of patients. We previously demonstrated that platelet reactivity is enhanced by a prothrombotic effect of erythrocytes in a thromboxane-independent manner. This diminishes the antithrombotic therapeutic potential of ASA. Recent data from our laboratory indicate that the prothrombotic effect of erythrocytes also contains an ASA-sensitive component. In accordance with this observation, intermittent treatment with high-dose ASA reduced the prothrombotic effects of erythrocytes ex vivo in healthy volunteers. In the present study, the effects of platelet-erythrocyte interactions were evaluated ex vivo in 82 patients with vascular disease: 62 patients with ischemic heart disease treated with 200 mg ASA/d and 20 patients with ischemic stroke treated with 300 mg ASA/d. METHODS AND RESULTS Platelet activation (release reaction) and platelet recruitment (fluid-phase proaggregatory activity of cell-free releasates from activated platelets) were assessed after collagen stimulation (1 microg/mL) of platelets, platelet-erythrocyte mixtures, or whole blood. Platelet thromboxane A2 synthesis was inhibited by >94% by ASA administration in all patients. Importantly, platelet recruitment followed one of three distinct patterns. In group A (n=32; 39%), platelet recruitment was blocked by ASA both in the presence and absence of erythrocytes. In group B (n=37; 45%), recruitment was abolished when platelets were evaluated alone but continued in the presence of erythrocytes, indicating a suboptimal effect of ASA on erythrocytes of this patient group. In group C (n= 13; 16%), detectable recruitment in stimulated platelets alone persisted and was markedly enhanced by the presence of erythrocytes. CONCLUSIONS In two thirds of a group of patients with vascular disease, 200 to 300 mg ASA was insufficient to block platelet reactivity in the presence of erythrocytes despite abolishing thromboxane A2 synthesis. Platelet activation in the presence of erythrocytes can induce the release reaction and generate biologically active products that recruit additional platelets into a developing thrombus. Insufficient blockade of this proaggregatory property of erythrocytes can lead to development of additional ischemic complications.

Superoxide production and reducing activity in human platelets.

Human platelets contain the cuprozinc (cytoplasmic) and manganese (mitochondrial) forms of superoxide dismutase. Nevertheless, superoxide radicals were detectable in the surrounding medium of metabolically viable platelet suspensions by using two assay systems: cytochrome c and nitroblue tetrazolium. The quantity of superoxide generated by platelets (5 X 10(5) superoxide radicals/platelet per 10 min) was constant and did not increase after aggregation by agents such as collagen and thrombin. The superoxide-generating system was present in the supernate of both aggregated and resting platelets and therefore was not platelet-bound. Platelet superoxide production was unaffected by prior ingestion of aspirin, indicating that the prostaglandin and thromboxane pathways were not involved. Both resting and aggregated platelets exhibited a reductive capacity toward cytochrome c and nitroblue tetrazolium which was unrelated to superoxide production. Furthermore, the aggregation process always resulted in a marked increase in this reduction. The nonsuperoxide reduction associated with aggregation was found to be membrane bound and to decrease with an apparent first order reaction rate (k1 = 0.067 min-1). In addition, accumulative, time-dependent nonsuperoxide-related cytochrome c reduction was also detected. Since there is no superoxide dismutase in plasma, the presence of superoxide radicals in the surrounding medium of platelets may have in vitro significance for platelet and leukocyte concentration and storage and in vivo significance for hemostasis, coagulation, and thrombosis. The nonsuperoxide-related reducing activities may represent a biochemical basis for platelet-blood vessel interactions, with particular reference to blood vessel integrity.

Inhibition of platelet function by an aspirin-insensitive endothelial cell ADPase. Thromboregulation by endothelial cells.

We previously reported that platelets become unresponsive to agonists when stimulated in combined suspension with aspirin-treated human umbilical vein endothelial cells. Inhibition occurred concomitant with metabolism of platelet-derived endoperoxides to prostacyclin by endothelial cells. We now demonstrate that if aspirin-treated platelets which fully respond to appropriate doses of agonists are exposed to aspirin-treated endothelial cells, they remain unresponsive despite absence of prostacyclin. Platelet inhibition is due in large part to ecto-ADPase activity on the endothelial cells. This was established by incubating aspirin-treated endothelial cells with 14C-ADP. Radio-thin layer chromatography and aggregometry demonstrated that 14C-ADP and induction of platelet activation decreased rapidly and concurrently. AMP accumulated transiently, was further metabolized to adenosine, and deaminated to inosine. The apparent Km of the endothelial cell ADPase was 33-42 microM and the Vmax 17-43 nmol/min per 10(6) cells, values in the range of antithrombotic potential. Thus, at least three complementary systems in human endothelial cells control platelet responsiveness: a cell-associated, aspirin-insensitive ADPase which functions in parallel with fluid phase autacoids such as the aspirin-inhibitable eicosanoids, and the aspirin-insensitive endothelium-derived relaxing factor.