Background-Platelet surface P-selectin is considered the "gold standard" marker of platelet activation. Degranulated, P-selectin-positive platelets, however, aggregate with leukocytes in vitro and rapidly lose surface P-selectin in vivo. Methods and Results-Flow cytometric tracking of autologous, biotinylated platelets in baboons enabled us to directly demonstrate for the first time in vivo that (1) infused degranulated platelets very rapidly form circulating aggregates with monocytes and neutrophils, and (2) 30 minutes after infusion of the degranulated platelets, the percentage of circulating monocytes aggregated with infused platelets persist at high levels, whereas the percentage of circulating neutrophils aggregated with infused platelets and the platelet surface P-selectin of nonaggregated infused platelets return to baseline. We therefore performed 2 clinical studies in patients with acute coronary syndromes. First, after percutaneous coronary intervention (nϭ10), there was an increased number of circulating monocyte-platelet (and to a lesser extent, neutrophil-platelet) aggregates but not P-selectin-positive platelets. Second, of 93 patients presenting to an Emergency Department with chest pain, patients with acute myocardial infarction (AMI) (nϭ9) had more circulating monocyteplatelet aggregates (34.2Ϯ10.3% [meanϮSEM]) than patients with no AMI (nϭ84, 19.3Ϯ1.4%, PϽ0.05) and normal control subjects (nϭ10, 11.5Ϯ0.8%, PϽ0.001). Circulating P-selectin-positive platelets, however, were not increased in chest pain patients with or without AMI.

To examine the hypothesis that surface Pselectin-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 nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin xllb183), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (Pselectin-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 preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h 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 PKIH2-labeled, P-selectinnegative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.P-selectin, a member of the selectin family which includes Eand L-selectin, is a cell-adhesion molecule of activated platelets and endothelial cells (1, 2). P-selectin (also known as CD62P, GMP-140, and PADGEM protein) is a component of the a granule membrane of resting platelets that is only expressed on the platelet surface membrane during and after platelet degranulation and secretion (1,2). A soluble form of P-selectin circulates in plasma (3).Platelet surface P-selectin mediates the adherence of degranulated platelets to leukocytes in vitro (4, 5) and in vivo (6). It has therefore been postulated that surface P-selectin-positive (degranulated) platelets may be rapidly cleared from the circulation by leukocytes (2,4,5,7,8). In apparent contradiction to this postulate, other investigators have reported that degranulated platelets are no more rapidly cleared from the circulation than control platelets (9, 10). Methods have not previously been available to directly address this issue.In this study, we used novel three-color whole blood flow cytometric methods for tracking of circulating degran...

IN THE LAST FEW YEARS, there has been great interest in the study of chaotic behavior in nonlinear systems. Particular interest has been focused on the description of system behavior during the transition from stable to chaotic regimes. One intriguing result has been the description of a "universal" period-doubling route to chaos, suggesting that a diverse set of complex dynamical systems approach their chaotic regimes through a universal pathway. 1-3 The touchstones along this common route to chaos are perioddoubling or subharmonic bifurcations. A system driven at a fixed frequency w begins to demonstrate

A method for the separation of platelets on the basis of their size has been developed using counterflow centrifugation. Platelets were separated, free of plasma proteins and other cells, into seven subpopulations. The smallest-sized platelets, designated as Fraction 1, had a mean platelet volume (MPV) of 3.94 +/- 0.60 micrometer 3 (SD). Each successive fraction had a progressively larger MPV. The MPV for the largest-sized platelets, designated Fraction 7, was 8.19 +/- 0.64 micrometer 3. The MPV for the original platelets prior to fractionation was 6.57 +/- 0.61 micrometer 3. The mean density of Fraction 1 platelets was 1.067 +/- 0.002 g/cm3, while Fraction 7 had a mean density of 1.072 +/- 0.001 g/cm3. Transmission electron microscopy demonstrated that Fraction 1 had 4.3 +/- 0.9 dense bodies per platelet, and Fraction 7 had 12.6 +/- 2.4 dense bodies per platelet. Platelet LDH activity showed that the Fraction 1 platelets had 4.77 +/- 0.92 iu per 10(10) platelets; Fraction 7 platelets had 14.88 +/- 1.23 iu per 10(10) platelets. The LDH activity in the platelets before separation into subpopulations was 9.47 +/- 1.45 iu per 10(10) platelets. Platelet function was measured by ADP-induced aggregation, serotonin uptake, and thrombin-induced release. Progressively more rapid and more complete aggregation was observed as the platelet size increased over the seven fractions. Serotonin uptake was 4.2 times greater in the Fraction 7 platelets than in the Fraction 1 platelets. Quantitative release of serotonin following thrombin stimulation was significantly greater in the larger-sized platelets than in the smaller-sized platelets. The observed differences in platelet aggregation, dense body content, LDH activity, and serotonin uptake and release suggest that large platelets may be functionally more important than smaller platelets.

(1) Cryopreserved platelet transfusions are superior to liquid-preserved platelets in reducing blood loss and the need for blood product transfusions after cardiopulmonary bypass. (2) The reduction in blood loss in the patients receiving cryopreserved platelet transfusions after cardiopulmonary bypass probably reflects improved in vivo hemostatic function of cryopreserved platelets. (3) Some in vitro measures of platelet quality (aggregation, pH, hypotonic stress) may not reflect in vivo quality of platelet transfusions after cardiopulmonary bypass, whereas other in vitro measures (platelet procoagulant activity and thromboxane) do.

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 reperfused. 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 the use of monoclonal antibodies which prevent neutrophil-endothelial adhesion, a prerequisite for injury.

Baboons that were subjected to systemic hypothermia at 32 C had an arm skin temperature of 27.3 C and bleeding time of 5.8 minutes. With local warming of the arm skin to 34 C, the bleeding time was 2.4 minutes. In normothermic baboons with arm skin temperature of 34.6 C, the bleeding time was 3.1 minutes. Local cooling of the arm skin to 27.6 C produced a bleeding time of 6.9 minutes. Increasing the skin temperature of the arm in hypothermic baboons to 38.9 C and in normothermic baboons to 40.1 C reduced bleeding times to 2.1 and 2.3 minutes, respectively. In both hypothermic and normothermic baboons there was a negative and significant correlation between the bleeding time and the arm skin temperature and the thromboxane B2 level in the shed blood obtained at the template bleeding time site. There was a significant positive correlation between the thromboxane B2 level in the shed blood and the arm skin temperature. Both in-vivo and in-vitro studies have shown that the production of thromboxane B2 by platelets is temperature-dependent, and that a cooling of skin temperature produces a reversible platelet dysfunction. Data also suggest that when a hypothermic patient bleeds without surgical cause, skin and wound temperature should be restored to normal before the administration of blood products that are not only expensive but may also transmit disease.

Patients with stable CAD have circulating activated platelets, circulating monocyte-platelet aggregates, increased platelet reactivity and an increased propensity to form monocyte-platelet aggregates.