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.

SummaryVery few studies have examined platelet function in very low birth weight (VLBW) preterm neonates, because of the relatively large volumes of blood required. In this study, platelet function in clinically stable VLBW neonates was examined by whole blood flow cytometry, which requires only 5 |jl1 of whole blood per assay. The following monoclonal antibodies were used: S12 (P-selectin-specific, reflecting a granule secretion), PAC1 (directed against the fibrinogen binding site exposed on the GPIIb-IIIa complex of activated platelets), F26 (directed against a conformational change in fibrinogen bound to the GPIIb-IIIa complex), and 6D1 (directed against the von Willebrand factor binding site on the GPIb-IX-V complex). VLBW neonates, like normal adults, did not have circulating activated platelets, as determined by the lack of binding of SI2, PAC1, and F26 in the absence of an added agonist. VLBW neonatal platelets were markedly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619 (a stable thromboxane A2 analogue), as determined by the extent of increase in the platelet binding of SI2, PAC1, and F26, and the extent of decrease in the platelet binding of 6D1. In summary, compared to adults, the platelets of VLBW neonates are markedly hyporeactive to thrombin, ADP/epinephrine and a thromboxane A2 analogue in the physiologic milieu of whole blood, as determined by: 1) the increase in platelet surface P-selectin; 2) the exposure of the fibrinogen binding site on the GPIIb-IIIa complex; 3) fibrinogen binding; and 4) the decrease in platelet surface GPIb. This platelet hyporeactivity may be a factor in the propensity of VLBW neonates to intraventricular hemorrhage. In addition to its previously defined use as a test of platelet hyperreactivity, the present study suggests that whole blood flow cytometry may be useful in the clinical assessment of platelet hyporeactivity.

Background Shear stress–induced platelet aggregation may initiate arterial thrombosis at sites of pathological blood flow. Shear stress–induced platelet aggregation is mediated by von Willebrand factor (vWf) binding to platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa. Tissue-type plasminogen activator (TPA) induces thrombolysis in coronary arteries through the local generation of plasmin. Plasmin also proteolyses GP Ib and plasma vWf. Methods and Results Because these effects could mitigate shear stress–induced platelet aggregation, we investigated the effect of fibrinolytic agents on platelet aggregation in response to a pathological shear stress of 120 dynes/cm 2 generated by a cone-and-platen rotational viscometer. Plasmin inhibited shear stress–induced aggregation of washed platelets, and this was associated with a decrease in GP Ib. TPA, at concentrations ≥2000 IU/mL, significantly inhibited shear stress–induced platelet aggregation of platelet-rich plasma without a decrease in platelet GP Ib. In plasma-platelet mixing experiments, we determined that the TPA effect was localized to plasma. Purified vWf multimer degradation by TPA (in the presence of exogenous plasminogen) was associated with the loss of the capacity of vWf to support shear stress–induced platelet aggregation. Conclusions These results demonstrate that TPA inhibits platelet aggregation in response to pathological shear stress by altering the multimeric composition of vWf. This effect of TPA on shear stress–induced platelet aggregation may contribute, along with fibrinolysis, to the therapeutic effect of TPA in restoring blood flow during acute coronary artery thrombosis.

SummaryPlatelet-derived microparticles (PDMP) appear to play a major role in the generation of procoagulant activity. In this study, we describe a novel flow cytometric method that allows direct evaluation of the procoagulant activity of PDMP and platelets in the physiological milieu of whole blood. The percent PDMP generated in response to calcium ionophore A23187 and calcium was increased in preterm neonates (67.5 ± 3.4%, mean ± S. E. M., n = 8, p <0.05) and term neonates (67.2 ± 2.7%, n = 7, p<0.05) compared with adults (49.5 ± 3.4%, n = 13). However, in preterm neonates A23187/calcium-induced binding of factor V/Va to PDMP and platelets (22.8 ± 5.6 fluorescence units) was markedly reduced (p <0.05) compared to term neonates (58.2 ± 7.2) and adults (50.6 ± 6.3). In preterm blood, A23187/calciuminduced binding of factor V/Va to PDMP and platelets returned to adult levels when: a) adult plasma, rather than autologous preterm neonatal plasma, was added; or b) factor V, but not factor VIII, was added to autologous preterm neonatal plasma. In summary: 1) We have developed a flow cytometric method for the direct detection of procoagulant PDMP and platelets in whole blood. 2) Compared to adults and term neonates, PDMP and platelets of preterm neonates bound markedly less factor V/Va (reflecting reduced procoagulant activity), because of a relative lack of factor V in preterm neonates. 3) This procoagulant defect in PDMP and platelets may contribute to the propensity of preterm neonates, but not term neonates, to intraventricular hemorrhage. 4) The percent PDMP does not necessarily reflect the degree of procoagulant activity of PDMP or platelets.