Noninvasive measurement of platelet kinetics in normal and hypertensive pregnancies.

OBJECTIVE: Our purpose was to determine platelet kinetics in pregnancy by means of noninvasive reticulated platelet counts and to examine in a pilot study whether increased reticulated platelet values were associated with preeclampsia and pregnancy-induced hypertension. STUDY DESIGN: Nulliparous women had blood samples drawn at enrollment (first prenatal visit) and at 28 and 36 weeks' gestation. The percent of reticulated platelets (an index of marrow platelet release correlating with increased thrombopoiesis), platelet-associated immunoglobulin, and serum antiplatelet antibody were measured and correlated with the clinical course for each patient. RESULTS: In 31 normal pregnancies the percent of reticulated platelets was never significantly higher than the values for normal nonpregnant women (5.8% +/- 2.2%) in spite of a significant decrease in platelet count by 36 weeks. By contrast, the percent of reticulated platelets in four women with preeclampsia rose significantly to 13.9% +/- 11.2% at 28 weeks, before the onset of clinical signs. No women had evidence of immune platelet destruction. CONCLUSION: In normal pregnancy the decline in platelet count is not accompanied by an increase in marrow platelet production, suggesting that the platelet decrease is dilutional without a compensatory thrombopoietic response or alternatively that thrombopoiesis is down-regulated during normal pregnancy. However, platelet production does increase before the onset of symptoms in a small number of women in whom preeclampsia or pregnancy-induced hypertension subsequently develops. These findings may justify a larger prospective study to determine whether noninvasive serial measurement of the percent of reticulated platelets can predict those pregnant women at risk for hypertension and preeclampsia.

Aspirin does not inhibit adenosine diphosphate-induced platelet alpha-granule release.

The involvement of metabolites of arachidonic acid in platelet-dense granule secretion and secondary platelet-platelet interactions is well characterized. However, their role in heterotypic interactions dependent on alpha-granule secretion is less well understood. Using platelet-surface expression of P-selectin as a marker of alpha-granule secretion, we have shown that: (1) aspirin treatment of platelets at doses that block dense granule secretion does not inhibit alpha-granule secretion to adenosine diphosphate (ADP); (2) synergism between epinephrine and ADP in the induction of P-selectin expression is similarly unaffected by aspirin; and (3) the ability of P-selectin to mediate adhesion of activated platelets to monocytes and polymorphonuclear lymphocytes in whole blood is also unchanged by aspirin treatment. To further explore the mechanisms responsible for platelet alpha-granule secretion, we have shown that inhibition of Na+/H+ exchange by either acidification of the extracellular medium or amiloride treatment blocked ADP-induced P-selectin expression. In contrast, incubation with the platelet lipoxygenase inhibitor 5,8,11-eicosatrynoic acid, by itself and with aspirin, did not decrease ADP-induced P-selectin expression. We conclude that platelet alpha-granule secretion in response to ADP is dependent on intact Na+/H+ exchange but is independent of the lipoxygenase- and cyclooxygenase-dependent metabolites of arachidonic acid.

Reticulated platelets in the evaluation of thrombopoietic disorders.

The laboratory diagnosis of immune platelet destruction has relied predominantly on the presence or absence of megakaryocytes in bone marrow. Recently, examination of peripheral blood platelets for high RNA content (reticulated platelets) or for elevated levels of platelet-associated IgG have been suggested as less invasive diagnostic tests. We used thiazole orange fluorescence labeling to determine the percentage of circulating reticulated platelets and two antibodies with different specificities directed against human IgG to measure platelet-associated IgG by flow cytometry in 59 patients with either immune thrombocytopenic purpura (n = 23) or chemotherapy-induced thrombocytopenia (n = 36). The percentage of reticulated platelets in patients with immune thrombocytopenia was significantly increased (38.6% +/- 27.4% [mean +/- 1 SD]), compared with patients receiving chemotherapy and normal subjects (7.2% +/- 3.3% and 2.9% +/- 2.2%, respectively). However, 17% of patients with immune thrombocytopenia had reticulated platelet values in the range observed for normal subjects and for patients with chemotherapy. Although one third of patients with immune thrombocytopenia had very high platelet IgG levels, the majority could not be distinguished from patients receiving chemotherapy solely on this basis. Combining the reticulated platelet determination with the IgG data did not improve the sensitivity or specificity of the reticulated platelet determination alone. We conclude that a flow cytometric assay for reticulated platelets is a better discriminant than flow-measured platelet IgG for diagnosing immune platelet destruction. We further postulate that the subset (17%) of patients with immune destruction who have relatively low percentages of reticulated platelets may represent patients with an inappropriately low thrombopoietic response.

Determination of the percentage of thiazole orange (TO)-positive, "reticulated" platelets using autologous erythrocyte TO fluorescence as an internal standard.

Determination of the percentage of thiazole orange (TO)-positive or "reticulated" platelets by flow cytometry has been advocated as an aid in the diagnosis of thrombocytopenic disorders. However, a reproducible method for determining control fluorescence and setting threshold values on a routine clinical basis has not been described. We used erythrocyte TO fluorescence in whole blood as an internal standard to set threshold markers for TO fluorescence of autologous, purified platelets. Since platelets have approximately a threefold higher TO fluorescence than erythrocytes, multiplying the erythrocyte threshold marker by 3 allowed a determination of the percentage of "reticulated" platelets in 29 normal controls. This value was 3.64% +/- 2.10% (S.D.) in men and 5.79 +/- 2.22% (S.D.) in women; there was a significant (P < 0.05) difference between normal men and women for this measurement. Applying this method to our earlier studies of normals and patients with immune thrombocytopenic purpura confirmed its ability to discriminate between these populations with similar sensitivity and specificity as previously reported. This method controls for fluctuations in TO labeling and fluorescence and does not require daily determination of a new normal control value.

Activation in stored platelet concentrates: correlation between membrane expression of P-selectin, glycoprotein IIb/IIIa, and beta-thromboglobulin release.

By using two distinct measurements of alpha-degranulation (surface P-selectin [alpha-granule membrane protein-140] expression and beta-thromboglobulin [beta-TG] release) and quantitation of glycoprotein (GP) IIb/IIIa surface density, stored platelet concentrates were evaluated to determine a) which method of measuring platelet alpha-granule release was more sensitive in detecting early platelet activation; b) whether Day 1 levels of activation predicted the extent of activation or cell lysis on Day 5 of storage; and c) whether changes in surface GPIIb/IIIa density were primarily dependent on platelet activation. By using samples from paired and unpaired units stored for 1, 3, and 5 days, four observations could be made. 1) A flow cytometric assay for the percentage of P-selectin-positive platelets was more sensitive for early detection of platelet activation than was measurement of beta-TG release. This finding was most likely due to enhanced sensitivity in detecting platelets that had undergone partial alpha-granule release. 2) Total P-selectin expression correlated with beta-TG release, which indicated that the extent of alpha-granule membrane fusion with the external platelet membrane was proportional to the amount of alpha-granule contents released into the supernatant. 3) All of the activation measurements on Day 1 predicted the activation values, but did not predict the degree of cell lysis (measured by lactate dehydrogenase discharge), on Day 5 of storage. 4) Surface GPIIb/IIIa density was increased on the subset of P-selectin-positive platelets as compared with the P-selectin-negative subset at all times during storage, but, within each subset, GPIIb/IIIa surface density did not significantly increase over the time of storage.

Cardiopulmonary bypass induces leukocyte-platelet adhesion.

Cardiopulmonary bypass (CPB) has been demonstrated to activate platelets, producing an increased number of circulating platelets that have undergone alpha-granule release and express granule membrane protein-140 (GMP-140) on their surface. In vitro, GMP-140 mediates activated platelet adhesion to neutrophils (PMN) and monocytes, causing the formation of leukocyte-platelet conjugates. Using a newly developed assay that measures the percentage of circulating leukocyte-platelet conjugates in whole blood, we studied 17 patients undergoing CPB and have determined that (1) monocyte-platelet conjugates increased significantly during CPB, from 18% +/- 1.5% to 44% +/- 4.5% (mean +/- SEM) by the end of CPB, while PMN-platelet conjugates increased only slightly and lymphocyte-platelet conjugates decreased; (2) the time course of the increase in monocyte- and PMN-platelet conjugates paralleled that of the increase in circulating activated platelets, as determined by the presence of surface GMP-140; and (3) monocyte activation, as assessed by increased surface expression of CD11b, showed a gradual increase similar to the increase in monocyte-platelet conjugates, while PMN surface CD11b peaked immediately after the start of CPB. We conclude that CPB, through increased platelet GMP-140 expression, causes formation of monocyte-platelet, and to a lesser extent, PMN-platelet conjugates. The activation of monocytes and PMN on CPB, as evidenced by CD11b expression, occurs with differing time courses.

Dynamics of leukocyte-platelet adhesion in whole blood.

The dynamics of leukocyte-platelet adhesion and platelet-platelet interaction in whole blood are not well understood. Using different platelet agonists, we have studied the whole blood kinetics of these heterotypic and homotypic interactions, the relative abilities of different leukocyte subsets to participate in platelet adhesion, and the ligands responsible for adhesion. When platelet aggregation was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide, thrombin stimulation of whole blood resulted in platelet expression of granule membrane protein 140 (GMP-140) and, simultaneously, a marked increase in the percentage of monocytes and neutrophils (PMN) binding platelets, as well as an increase in the number of platelets bound per monocyte and PMN. Lymphocytes were unaffected. Monocytes bound more platelets and at an initially faster rate than PMN. This increase in monocyte and PMN adhesion to platelets was completely inhibited by the blocking monoclonal antibody (MoAb), G1, to GMP-140. When the combination of epinephrine and adenosine diphosphate (epi/ADP) was used as a less potent agonist in the presence of RGDS, GMP-140 expression per platelet was less, and while monocyte-platelet conjugates formed, PMN-platelet conjugates did not. With epi/ADP in the absence of RGDS, there was an immediate, marked decrease in the percentage of all leukocytes with bound platelets, simultaneous with an increase in the percentage of unbound platelet aggregates. As these platelet aggregates dissociated, the percentage of monocytes and PMN with adherent platelets increased, with monocytes again binding at a faster initial rate than PMN. This recovery of monocyte and PMN adhesion to platelets was also inhibited by the G1 MoAb. We conclude that: (1) monocytes and PMN bind activated platelets in whole blood through GMP-140; (2) monocytes have a competitive advantage over PMN in binding activated platelets, particularly when less potent platelet agonists are used; and (3) platelet aggregate formation initially competes unactivated platelets off leukocytes; subsequent aggregate dissociation allows the now activated platelets to readhere to monocytes and PMN through GMP-140. These studies further elucidate the dynamic interaction of blood cells and possible links between coagulative and inflammatory processes.

Activated and unactivated platelet adhesion to monocytes and neutrophils.

To examine the possible receptor-ligand pairs mediating adhesion of activated and "unactivated" platelets to leukocytes and the kinetics of leukocyte-platelet binding, we developed a flow cytometric assay using isolated cell fractions to accurately measure heterotypic cell adhesion, including both total leukocyte-platelet conjugate formation as well as the number of platelets bound per leukocyte. We have shown that (1) activated platelet binding to both polymorphonuclear leukocytes (PMN) and monocytes is dependent on both a specific epitope (blocked by monoclonal antibody G1) of granule membrane protein-140 (GMP-140) and the presence of divalent cations; (2) unactivated platelets bind to 87% of viable, resting monocytes but to only 34% of PMN; (3) the receptor(s) on unactivated platelets that mediate adhesion to PMN and monocytes do not require divalent cations and become nonfunctional after thrombin activation; and (4) the kinetics of platelet adhesion to monocytes and PMN indicate that monocyte adhesion is favored over neutrophil adhesion. We conclude that platelet-heterotypic cell adhesion is a dynamic process reflecting the activation status of the platelet and differential binding abilities of leukocytes.