M. Johan Broekman

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.

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.

Quantification of human platelet inositides and the influence of ionic environment on their incorporation of orthophosphate-32P

Platelets are a rich source for the study of inositol lipids in man. The substitution of an EDTA-KCl solution for the water component of the Bligh and Dyer procedure permitted quantitative extraction of polyphosphoinositides. The latter, with monophosphoinositide, were found to comprise, on a molar basis, 6.7% of total platelet phospholipids. Study of the incorporation of orthophosphate-(32)P into platelet phospholipids was further simplified by separating eight (32)P-labeled lipids, including the inositides, with a single chromatographic development on formaldehyde-treated paper. Particular attention was paid to the influence of ionic environment on the pattern and degree of labeling. In 300 mOsm media major phospholipids other than the inositides were not labeled. Small amounts of label appeared in certain trace phospholipids, notably phosphatidic acid. In 150 mOsm media, labeling of inositides was moderately increased, that of trace phospholipids enormously so. The increased labeling was not solely due to thrombocytolysis since (a) platelet disruption by sonication or freeze-thawing abolished (32)P incorporation into phospholipids and (b) in timed studies, restoration of osmolarity to 300 mOsm by addition of hypertonic sorbitol blunted the enhancement effect of previous 150 mOsm exposure. Lowering K and compensatorily increasing Na concentration of 300 mOsm media also stimulated (32)P labeling of inositides and, to a lesser extent, the trace phospholipids. However, the pattern and degree of stimulation were not as strikingly altered as in the osmolarity studies. These data show that drastic alterations of ionic environment can sharply influence the platelets ability to incorporate orthophosphate-(32)P into its phospholipids.

Distribution of Fibrinogen, and Platelet Factors 4 and XIII in Subcellular Fractions of Human Platelets

The distribution of fibrinogen, platelet factor 4 (heparin‐neutralizing factor) and factor XIII amongst subcellular fractions of human platelets was determined. It was found that fibrinogen and platelet factor 4 peaked sharply in the region of the density gradient previously shown to be heavily enriched with α‐granules. By contrast, factor XIII, fibrin‐stabilizing factor, was found exclusively in the supernatant.

Participation of Tyrosine Phosphorylation in Cytoskeletal Reorganization, αIIbβ3 Integrin Receptor Activation, and Aspirin-Insensitive Mechanisms of Thrombin-Stimulated Human Platelets

BackgroundFibrinogen binding to the active conformation of the &agr;IIb&bgr;3 integrin receptor (glycoprotein IIb/IIIa) and cytoskeletal reorganization are important events in platelet function. Tyrosine phosphorylation of platelet proteins plays an essential role in platelet signal transduction pathways. We studied the participation of tyrosine kinases on these aspects of platelet reactivity and their importance in cyclooxygenase (COX)-1–independent mechanisms in thrombin-stimulated human platelets. Methods and ResultsUsing washed platelets from normal donors and tyrphostin-A47 and aspirin as tyrosine kinase and COX-1 inhibitors, respectively, we found that tyrphostin-A47 downregulated (1) the thrombin-activated conformational change of &agr;IIb&bgr;3, (2) actin polymerization and cytoskeletal reorganization, and (3) the quantity of tyrosine-phospho-rylated proteins associated with the reorganized cytoskeleton. The latter are important components of multimolecular signaling complexes. Concomitantly, platelet aggregation and secretion were significantly reduced. Aspirin did not affect receptor activation or tyrosine phosphorylation but did decrease the initial (30-second) burst of actin polymerization. Importantly, aspirin significantly amplified the inhibitory effect of tyrphostin-A47 on all aspects of platelet reactivity that we evaluated. ConclusionsTyrosine protein phosphorylation is a regulatory control system of the inside-out mechanism of &agr;IIb&bgr;3 activation and cytoskeletal assembly in thrombin-stimulated human platelets. Inhibition of these aspects of platelet function with tyrphostin-A47 is amplified when platelets are treated with aspirin. Therefore, tyrosine phosphorylation is a major component of early signaling events and of COX-1–independent mechanisms of thrombin-induced platelet reactivity. The study results may indicate a novel target for therapeutic intervention.

Feedback Regulation of Endothelial Cell Surface Plasmin Generation by PKC-dependent Phosphorylation of Annexin A2

In response to blood vessel injury, hemostasis is initiated by platelet activation, advanced by thrombin generation, and tempered by fibrinolysis. The primary fibrinolytic protease, plasmin, can be activated either on a fibrin-containing thrombus or on cells. Annexin A2 (A2) heterotetramer (A2·p11)2 is a key profibrinolytic complex that assembles plasminogen and tissue plasminogen activator and promotes plasmin generation. We now report that, in endothelial cells, plasmin specifically induces activation of conventional PKC, which phosphorylates serine 11 and serine 25 of A2, triggering dissociation of the (A2·p11)2 tetramer. The resulting free p11 undergoes ubiquitin-mediated proteasomal degradation, thus preventing further translocation of A2 to the cell surface. In vivo, pretreatment of A2+/+ but not A2−/− mice with a conventional PKC inhibitor significantly reduced thrombosis in a carotid artery injury model. These results indicate that augmentation of fibrinolytic vascular surveillance by blockade of serine phosphorylation is A2-dependent. We also demonstrate that plasmin-induced phosphorylation of A2 requires both cleavage of A2 and activation of Toll-like receptor 4 on the cell surface. We propose that plasmin can limit its own generation by triggering a finely tuned “feedback” mechanism whereby A2 becomes serine-phosphorylated, dissociates from p11, and fails to translocate to the cell surface.

Targeted Deletion of Ectonucleoside Triphosphate Diphosphohydrolase 1/CD39 Leads to Desensitization of Pre- and Postsynaptic Purinergic P2 Receptors

We previously reported that ATP coreleased with norepinephrine from cardiac sympathetic nerves activates presynaptic P2X purinoceptors (P2XR), thereby enhancing norepinephrine exocytosis. Blockade of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1/CD39) potentiates norepinephrine exocytosis, whereas recombinant soluble CD39 (solCD39) in-hibits it. This suggested that CD39 gene (Entpd1) deletion would enhance purinergic and adrenergic signaling by preserving ATP and its norepinephrine-releasing activity. However, we found that the neurogenic contractile response of vasa deferentia from Entpd1-null (CD39–/–) mice was attenuated and accompanied by reduced activity of pre- and postsynaptic P2XR, whereas contractile responses to K+ or norepinephrine remained intact. In addition, the magnitude of ATP and norepinephrine exocytosis from cardiac synaptosomes was decreased in CD39–/– mice. Inhibition of E-NTPDase1/CD39, or solCD39 administration, did not affect the attenuated contractile response of vasa deferentia from CD39–/– mice. Notably, Entpd1 deletion and pharmacological P2XR desensitization in control mice similarly attenuated vasa deferentia responses. Thus, excessive and prolonged ATP exposure resulting from CD39 deletion desensitizes pre- and postjunctional P2XR at the sympathetic neuromuscular junction. This diminishes purinergic activity directly and adrenergic activity indirectly. It remains to be determined whether this desensitization results from receptor internalization, changes in receptor conformation or phosphorylation. Shutdown of ATP signaling in CD39–/– mice may represent a defense mechanism for the prevention of purinergic overstimulation. Our findings emphasize the cardioprotective role of neuronal CD39: by reducing presynaptic facilitatory effects of neurotransmitter ATP, CD39 attenuates norepinephrine release and its dysfunctional consequences. Moreover, by virtue of its antithrombotic action CD39 can potentially prevent the transition from myocardial ischemia to infarction.