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To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from non-human primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and re-infused into the same baboons. Three color whole blood flow cytometry was used to simultaneously: (1) identify platelets with a monoclonal antibody directed against GPIIb-IIIa (integrin alpha sub IIb Beta sub 3), (2) distinguish infused platelets by their PKH2 fluorescence, and (3) analyze platelet function with monoclonal antibodies. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin pre-infusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 hours after infusion and only slightly less 48 hours after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/mL increase in the plasma concentration of soluble P selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their: (a) participation in platelet aggregates emerging from a bleeding time wound, (b) binding to Dacron in an arteriovenous shunt, (c) binding of monoclonal antibody PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (d) generation of procoagulant platelet-derived microparticles.

Ischaemia is a common clinical event leading to local and remote injury. Evidence indicates that tissue damage is largely caused by activated neutrophils which accumulate when the tissue is perfused. If the area of ischaemic tissue is large, neutrophils also sequester in the lungs, inducing non-cardiogenic pulmonary oedema. Ischaemia-reperfusion injury is initiated by production of reactive oxygen species which initially appear responsible for the generation of chemotactic activity for neutrophils. Later, once adherent to endothelium, neutrophils mediate damage by secretion of additional reactive oxygen species as well as proteolytic enzymes, in particular elastase. Therapeutic options for limiting ischaemia-reperfusion injury include inhibition of oxygen radical formation, pharmacological prevention of neutrophil activation and chemotaxis, and also use of monoclonal antibodies which prevent neutrophil-endothelial adhesion, a prerequisite for injury.

Prostacylin (PGI2) productions closely coupled with endothelial cell shape and F-actin distribution in vitro. These findings may implicate cytoskeletal constituents in a mechanism regulating eicosanoid metabolism. To determine the potential for such a regulatory mechanism, cytoskeletal protein effects on the rate-limiting eicosanoid cascade enzyme (phospholipase A2, PLA2) were studied. Membrane phospholipid degradation was indirectly determined by spectrophotometric measurement of PLA 2 induced rat red blood cell ghost (RBC-G) hemolysis. PLA 2 was incubated with actin (skeletal, smooth, or non-muscle cell) at a non-muscle cell concentration (100 micromoles and then exposed to the RBC-G Comparisons in the presence or absence of actin revealed that F-actin stimulated whereas G-action suppressed PLA2 lytic behavior significantly (P< 0.05). When a 10: or 100: 1 F-actin to myosin-ratio was used, the F-actin stimulatory effect was significantly (P<0.05) reduced. These finding s suggest that the in vitro correlation between PGI 2 production and endothelial cell shape may be the result of PLA 2 regulation by cytoskeletal elements that impart cellular form.

Alterations in endothelial cell shape, as measured by the planar surface area encompassed by the cell perimeter adhering to the substrate, were associated with modulation of basal prostacyclin synthesis. Cells with small surface area generated the highest levels of prostacyclin and had both a diffuse arrangement of F-actin and an increased binding capacity for rhodamine-phalloidin. Large cells with many actin cables and a significantly reduced phalloidin binding capacity produced significantly smaller quantities of prostacyclin. These findings suggest a cytoskeletal role in the regulation of prostacyclin production and more specifically, that changes in F-actin arrangement modulate basal endoperoxide metabolism.

The general objectives of this laboratory have been to study cardiopulmonary abnormalities associated with shock and trauma, with a focus on the intermediary role of lung and circulating blood cell metabolism. Pressure breathing, pulmonary embolism and acid aspiration, by altering arachidonic acid metabolism were found to depress cardiac contractility and alter the distribution of systematic blood flow. The roles of prostacyclin (PG12) and thromboxane (Tx) A2 in these settings were quantitated by radioimmunoassay and use of selective antagonists. These same prostanoids, along with platelet serotonin, also moderated changes in pulmonary function, particularly physiologic shunt, physiologic dead space and pulmonary vascular resistance. The secondary consequence of injury, the recruitment of an inflammatory reaction, was found to be a significant determinant of increased microvascular permeability both in acid injury of the lungs and heat injury to the skin. In both instances, neutrophil accumulations and permeability edema could be attenuated by inhibition of Tx synthesis. These data indicate that the prostanoids exert direct and indirect actions in moderating cardiopulmonary function following injury.

Endothelial cell prostacyclin (Phosphogluculsomerase) production is closely coupled with cell shape. Since shape change implies cytoskeletal modulations, this finding may be an important clue to implicate cytoskeletal constituents in a mechanism regulating eicosanoid metabolism. Endothelial cells with a diffuse F-actin distribution generate more PGI than cells with many discrete F-actin stress fibers. Because actin can modulate enzyme activity, the myofibril protein effects on phopholipase A(PLA), the rate limiting eicosanoid cascade enzyme, were studied. F-actin stimulated whereas G-actin suppressed enzyme activity. Myosin in the presence of F-actin, the F-action stimulatory effect was significantly reduced. These findings suggest that the correlation of endothelial cell PGI metabolic modulation to cell shape and actin distribution was perhaps due to changes in PLA activity as a direct result of alterations in the polymerized state of actin and the degree of actin-myosin stress fiber formation.

Pressure breathing and particularly end-expiratory pressure will induce the pulmonary production of prostaglandins (PG). These PGs regulate cardiac contractility and the release of plasmin mediated fibrinolytic activity. A high molecular weight protein has been tentatively identified as the circulating negative inotropic agent whose production is stimulated by PG synthesis during end-expiratory pressure. The large amount of prostacyclin (PGI2) secreted by the lungs in response to surgery may protect against microaggregate entrapment and damage of the lungs. PGI2 infusion is effective therapy for experimental pulmonary embolism.

Investigations are continuing into the relationship of pulmonary prostaglandin production and systemic organ function. Our study preparations are of pulmonary embolism and/or positive end-expiratory pressure (PEEP) which we believe to be analogous to the microembolism and hyperventilation of severe injury. Both embolism and PEEP lead to the production of thromboxanes (Tx) as well as decreases in cardiac contractility. The latter event can be prevented with Tx antagonists. Tx cause the elaboration of a high molecular weight protein fraction which suppresses myocardial Ca(++)-ATPase and reduces activity of Krebs cycle enzymes in cardiac mitochondria. During acute thrombocytopenia serotonin (5-HT) infusion was found to protect against petechiae. Antagonists to 5-HT promoted petechiae. Endogenous or exogenously infused prostacyclin (PGI2) caused a reduction in plasma and increase in platelet 5-HT. Endothelial 5-HT transport was blocked. These findings may underly the ability of PGI2 to enhance permeability. Prostacyclin was found to regulate the production of plasminogen activator. This may be one of the mechanism related to the dramatic effectiveness of PGI2 in reversing the cardiopulmonary abnormalities of pulmonary embolism and in reversing lethal endotoxemia.