Background. It has been hypothesized that platelets are activated, or made more activatible, by strenuous exercise and that these changes may play a role in the genesis of exercise-induced coronary ischemia. Previous studies have yielded conflicting results but have used assays (eg, platelet aggregation, plasma platelet factor 4, and plasma (-thromboglobulin) that are subject to methodological problems.Methods and Results. In the present study, a whole blood flow cytometric method was used to study the

In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.

The use of cardiopulmonary bypass (CPB) during cardiac surgery is associated with a hemostatic defect, the hallmark of which is a markedly prolonged bleeding time. However, the nature of the putative platelet function defect is controversial. In this study, blood was analyzed at 10 time points before, during, and after CPB. We used a whole-blood flow cytometric assay to study platelet surface glycoproteins in (1) peripheral blood, (2) peripheral blood activated in vitro by either phorbol myristate acetate, the thromboxane (TX)A2 analog U46619, or a combination of adenosine diphosphate and epinephrine, and (3) the blood emerging from a bleeding-time wound (shed blood). Activation-dependent changes were detected by monoclonal antibodies directed against the glycoprotein (GP)Ib-IX and GPIIb-IIIa complexes and P-selectin. In addition, we measured plasma glycocalicin (a proteolytic fragment of GPIb) and shed-blood TXB2 (a stable breakdown product of TXA2). In shed blood emerging from a bleeding-time wound, the usual time-dependent increase in platelet surface P-selectin was absent during CPB, but returned to normal within 2 hours. This abnormality paralleled both the CPB-induced prolongation of the bleeding time and a CPB-induced marked reduction in shed-blood TXB2 generation. In contrast, there was no loss of platelet reactivity to in vitro agonists during or after CPB. In peripheral blood, platelet surface P-selectin was negligible at every time point, demonstrating that CPB resulted in a minimal number of circulating degranulated platelets. CPB did not change the platelet surface expression of GPIb in peripheral blood, as determined by the platelet binding of a panel of monoclonal antibodies, ristocetin-induced binding of von Willebrand factor, and a lack of increase in plasma glycocalicin. CPB did not change the platelet surface expression of the GPIIb-IIIa complex in peripheral blood, as determined by the platelet binding of fibrinogen and a panel of monoclonal antibodies. In summary, CPB resulted in (1) markedly deficient platelet reactivity in response to an in vivo wound, (2) normal platelet reactivity in vitro, (3) no loss of the platelet surface GPIb-IX and GPIIb-IIIa complexes, and (4) a minimal number of circulating degranulated platelets. These data suggest that the “platelet function defect” of CPB is not a defect intrinsic to the platelet, but is an extrinsic defect such as an in vivo lack of availability of platelet agonists. The near universal use of heparin during CPB is likely to contribute substantially to this defect via its inhibition of thrombin, the preeminent platelet activator.

SummaryPrevious studies have reported that the platelets of healthy term neonates have either diminished or normal reactivity compared to the platelets of adults. To circumvent the methodologic problems of previous studies, we used a whole blood flow cytometric method to study neonatal platelet reactivity to thrombin, a combination of ADP and epinephrine, and U46619 (a stable thromboxane A2 analogue). Inclusion in the assay of the peptide GPRP (an inhibitor of fibrin polymerization) enabled us to study platelet reactivity to human α-thrombin in whole blood. Umbilical cord blood and day 1 peripheral blood were collected from 30 healthy term neonates and compared to peripheral blood from 20 normal adults. In whole blood samples without added agonist, there were no significant differences between neonates and adults in the platelet binding of monoclonal antibodies 6D1 (GPIb-specific) or 7E3 (GPIIb-IIIa complex-specific). As determined by S12 (a P-selectin-specific monoclonal antibody), neither neonates nor adults had circulating degranulated platelets. However, in both cord and peripheral whole blood samples, neonatal platelets were significantly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619, as determined by the extent of increase in the platelet surface expression of P-selectin and the GPIIb-IIIa complex, and the extent of decrease in the platelet surface expression of the GPIb-IX complex. For example, as compared to maximal platelet surface P-selectin in adults (with thrombin 10 U/ml), thrombin 1 U/ml resulted in platelet surface P-selectin of 95 ± 2% (mean ± S.E.M.) in adult peripheral blood, but only 70 ± 4% in cord blood and 70 ± 3% in neonatal peripheral blood (p <0.0001). Thrombin 0.1 U/ml resulted in platelet surface P-selectin of 49 ± 4% in adult peripheral blood, but only 10 ± 2% in cord blood and 17 ± 2% in neonatal peripheral blood (p α0.0001). Similar results were obtained in a washed platelet system. In summary: 1) Compared to adult controls, neonatal platelets are hyporeactive to thrombin, a combination of ADP and epinephrine, and a thromboxane A2 analogue in the physiologic milieu of whole blood. 2) The hyporeactivity of neonatal platelets compared to adult platelets is the result of a defect intrinsic to neonatal platelets. 3) Whole blood flow cytometry is particularly advantageous for neonatal studies because only 5 μl of blood per assay is required.

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