A Comparison of Platelet Production in Mice made Thrombocytopenic by Hypoxia and by Platelet Specific Antisera

S ummary . Previous experiments have shown that hypoxia causes thrombocytopenia in mice. In an attempt to define the recovery phase, mice were enclosed in hypoxia chambers for 14 d and platelet counts, total circulating platelet counts (TCPC) and masses (TCPM), platelet sizes, and %³⁵S incorporation into platelets were determined over the next 8 d while the mice were kept at ambient O₂ levels. For comparison, untreated mice and mice made thrombocytopenic by injection of rabbit anti-mouse-platelet serum (RAMPS) were used. Both 14 d of hypoxia and RAMPS treatment resulted in severe thrombocytopenia. TCPC and TCPM were similar in both exhypoxic and RAMPS-injected mice. However, the platelet count recovery pattern of exhypoxic-thrombocytopenic mice did not show the rebound-thrombocytosis which was characteristic of RAMPS-induced thrombocytopenia, apparently because of dilution of platelets by increased blood volumes. Average platelet sizes were larger than normal 2 d posthypoxia or after RAMPS-treatment, followed by a decline toward normal; platelets from exhypoxic mice, however, remained larger. Per cent ³⁵S incorporation into platelets was lower in exhypoxic mice than in RAMPS-treated mice; lower numbers of megakaryocytes were observed immediately after removal from hypoxia followed by an increase in number and size by 2 d later. Also, thrombopoietin was detected in plasma of RAMPS-treated mice, but not in plasma of exhypoxic-thrombocytopenic mice. Therefore, it seems possible that hypoxia reduced the numbers of megakaryocytes, resulting in depressed thrombocytopoiesis of mice at the time of removal from hypoxic atmospheres.     

A Comparison of Platelet Size, Platelet Count, and Platelet 35S Incorporation as Assays for Thrombopoietin

SUMMARY. Average platelet size, platelet count, and ³⁵S-incorporation into platelets were compared as methods for the measurement of thrombopoietin-stimulated thrombopoiesis. In mice injected with rabbit anti-mouse platelet serum (RAMPS) average platelet size was shown to be increased as mice were recovering from thrombocytopenia. Also, ³⁵S-measurements on platelets of these mice showed significant increases in cpm/average platelet 2–4 days after RAMPS treatment. Significant increases in ³⁵S-incorporation into the total circulating mass of platelets were found on days 3–4. In normal mice or mice in rebound-thrombocytosis injected with thrombopoietin, platelet size remained unchanged, whereas the platelet count and ³⁵S-incorporation into platelets were shown to be significantly increased. Moreover, a dose-response experiment in mice pretreated with RAMPS showed a slight increase in platelet count as the dose of TSF was increased, but platelet sizes were unaltered. The %³⁵S-incorporation into platelets showed a significant linear dose-response, i.e. as the dose of thrombopoietin was increased, an increase in %³⁵S-incorporation into platelets was observed. These data indicated that of the three indirect measurements of thrombopoietin, the %³⁵S-incorporation into mouse platelets was the most sensitive, followed by platelet counting; the least sensitive measurement of thrombopoiesis was change in platelet size.     

Impaired platelet reactivity in patients with septic shock: a proof-of-concept study

Abstract

Coagulation disorders and thrombocytopenia are common in patients with septic shock, but only few studies have focused on platelet variables beyond platelet count. The aim of this study was to evaluate whether platelets reactivity predicts sepsis-induced thrombocytopenia in patients with septic shock.

We therefore enrolled consecutive patients with septic shock and platelets count >150*10³/μL on the day of the diagnosis. Platelets reactivity tests were performed daily from the diagnosis of septic shock until day five; platelet volume distribution and mean platelet volume were also recorded daily. Sepsis-induced thrombocytopenia was defined as a platelet count <150*10³/μL.

Thirty patients were included; sepsis-induced thrombocytopenia occurred in 11 (31%) patients. Platelets reactivity and platelet count at day of septic shock diagnosis were not correlated. Patients who experienced thrombocytopenia had lower maximal aggregation at diagnosis than others. Maximal aggregation tests were predictors of thrombocytopenia (AUROC from 0.756 to 0.797, depending on the agonist used). Both platelet volume distribution width and mean platelet volume were predictors of 90-day mortality (AUROC 0.866 and 0.735, respectively).

In this pilot study, impaired platelets reactivity was more common in patients who subsequently developed sepsis-induced thrombocytopenia; also, platelet volume distribution width and mean platelet volume were predictors of 90-day mortality.     

Tissue uptake of circulating thrombopoietin is increased in immune-mediated compared with irradiated thrombocytopenic mice

We have previously demonstrated a significant inverse correlation between circulating thrombopoietin (TPO) levels and peripheral platelet (PLT) counts in patients with thrombocytopenia secondary to megakaryocytic hypoplasia but not in patients with immune thrombocytopenic purpura (ITP; Chang et al, Blood 88:3354, 1996). To test the hypothesis that the differences in the circulating TPO levels in these two types of thrombocytopenia are caused by differences in the total capacity of Mpl receptor-mediated TPO clearance, thrombocytopenia was induced in female CD-1 mice either by sublethal irradiation (irradiated) or rabbit antimouse PLT serum (RAMPS) for 1 day (1 d RAMPS) and 5 days (5 d RAMPS). A well-characterized murine model of autoimmune thrombocytopenic purpura, male (NZW x BXSB) F1 mice (W/B F1), was also included in this study. All thrombocytopenic mice and their controls received trace amounts of 125I-recombinant murine TPO (125I-rmTPO) intravenously and were killed 3 hours postinjection. Blood cell-associated radioactivity was significantly decreased in all 4 groups of thrombocytopenic mice. Significantly increased plasma and decreased whole spleen-associated radioactivity was observed in the irradiated group compared with controls (P <.05). While a lesser but still significant increase in plasma and decrease in whole spleen-associated radioactivity was observed in the 1 d RAMPS mice (P <.05), there were no significant differences between the 5 d RAMPS nor the W/B F1 male mice compared with controls, although whole spleen-associated radioactivity was higher in the W/B F1 male. A significant inverse correlation of plasma and whole spleen-associated radioactivity was demonstrated in W/B F1 male mice (r = -.91, n = 6, P <.05). There was also a decrease in bone (femur)/blood-associated radioactivity in the irradiated group compared with controls (P <.05), but a significant increase in 1 d and 5 d RAMPS mice (P <.01). Furthermore, the 125I-rmTPO uptake capacity within the spleen and marrow of immune thrombocytopenic mice appeared to be associated with a higher megakaryocytic mass when tissue samples were examined by light microscopy. Internalization of 125I-rmTPO by megakaryocytes and PLTs in the spleens and marrows of ITP mice was also demonstrated directly using electron microscopic autoradiography. Labeled PLTs were also found within splenic macrophages. Additionally, the mean PLT volumes of RAMPS mice were significantly higher than those of the control and irradiated mice (P <.05), as was the bound 125I-rmTPO (cpm) per million PLT (P <.05). Finally, significantly decreased 125I-rmTPO degradation products were only found in the plasma of the irradiated mice compared with control animals (P <.05). These data suggest that the lack of Mpl+ cells in the mice with thrombocytopenia secondary to megakaryocytic hypoplasia (irradiated) results in decreased uptake and degradation of TPO and higher circulating TPO levels. Furthermore, these data also suggest that, after a brief TPO surge in response to immune thrombocytopenia (1 d RAMPS), the lack of an inverse correlation of circulating TPO with PLT counts during steady-state immune thrombocytopenic mice (5 d RAMPS + W/B F1 male) is due, at least in part, to its uptake and degradation by the high PLT turnover and increased mass of megakaryocytes.     

Thrombopoietin derived from human embryonic kidney cells stimulates an increase in DNA content of murine megakaryocytes in vivo

A thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) is known to increase the size and number of mouse bone marrow megakaryocytes, to increase the proportion of megakaryocytes in endomitosis, and to increase the number of small acetylcholinesterase-positive cells. Megakaryocyte ploidy values have not previously been measured in mice treated with TSF from human embryonic kidney (HEK) cells. Therefore, in the present study C3H mice were injected with approximately 4 U of step II TSF; platelet production indices and megakaryocyte ploidy values were measured 1-5 days after treatment. For controls, other mice were injected with saline, human serum albumin (HSA), normal rabbit serum (NRS), or rabbit anti-mouse platelet serum (RAMPS). Platelet counts, platelet sizes, and percent 35S incorporation into platelets were measured using standard techniques. For measurement of megakaryocyte DNA content, bone marrow cells were collected into CATCH medium and incubated with RAMPS, followed by labeling with a saturating concentration of fluorescein-conjugated goat anti-rabbit immunoglobulin F(ab')2 fragment. After washing, the cells were resuspended in propidium iodide, and DNA content was measured by flow cytometry. When compared to suitable control values, the results showed that TSF caused a significant (p less than 0.025) increase in platelet counts of treated mice by 3 days; both TSF and RAMPS caused significant (p less than 0.0005) increases in platelet sizes and percent 35S incorporation into platelets of mice at 2 and 3 days after treatment. The most frequent polyploid DNA class of megakaryocytes from untreated C3H mice was 32N, confirming our previous observation. Both TSF and RAMPS caused significant (p less than 0.0005) increases in the average polyploid megakaryocyte DNA content, with peak values on days 2 and 3. These data show that TSF administered in vivo significantly increases DNA content of mouse bone marrow megakaryocytes.     

Thrombin-induced increase in surface expression of epitopes on platelet membrane glycoprotein IIb/IIIa complex and GMP-140 is a function of platelet age

Platelets are heterogeneous in the content of membrane glycoprotein (GP)IIb/IIIa complex. To determine whether this heterogeneity is related to changes associated with platelet aging in the circulation, newly released platelets, obtained during recovery from nonimmune- mediated acute experimental thrombocytopenia in baboons, were studied. Monoclonal antibody (MoAb) binding to epitopes expressed on GPIIb/IIIa complex (LJ-CP8), GMP-140 (S12), and GPIa/IIa (12F1) was measured on control platelets (comprising platelets with a normal age distribution; mean age 60 to 72 hours) and newly formed platelets (mean age 12 hours), both in the resting state and after thrombin stimulation. Whereas LJ-CP8 binding to resting control platelets increased by 34% upon stimulation by gamma-thrombin from 30,885 +/- 1,171 to 41,458 +/- 1,311 molecules/platelet at saturating concentrations of antibody, LJ- CP8 binding to resting young platelets did not increase significantly upon thrombin stimulation (31,878 +/- 3,330 and 33,791 +/- 3,486 molecules/platelet, respectively). Similarly, binding of antibody S12 in response to maximal thrombin stimulation was reduced by 42% from 10,246 +/- 834 molecules/platelet at saturating concentrations of S12 for control platelets to 5,971 +/- 665 molecules/platelet for young platelets (P = .001). S12 binding to unstimulated platelets was less than 10% of the binding observed after thrombin stimulation at all concentrations of S12 for both control and young platelets. However, maximal binding of antibody 12F1 to resting control platelets did not differ significantly from that observed with resting young platelets (2,926 +/- 167 and 2,857 +/- 208 molecules/platelet, respectively), and 12F1 binding was unchanged after thrombin stimulation for both control and young platelets. We conclude that the thrombin-induced increase in the expression of epitopes on platelet membrane GPIIb/IIIa complex and GMP-140 is a function of platelet age.     

Platelets in idiopathic thrombocytopenic purpura are increased in size but are of normal density

To determine whether platelet size and volume are related to one another or to platelet age, subpopulations of platelets from patients with idiopathic thrombocytopenic purpura (ITP) have been produced on the basis of density using Percoll gradients. The density distribution of platelets from patients with ITP and from patients with other forms of thrombocytopenia (thought to be nonimmune in nature) was the same as in normal controls. However, the platelets in each density subpopulation from ITP patients were increased in size. beta-thromboglobulin (beta TG) content of platelets from each patient group and the normals increased with density and tended to be higher in ITP than in normal controls. beta TG concentration per unit platelet volume and its level in plasma were similar in ITP patients and in normal controls. This suggests that the apparently normal density of ITP platelets was not a result of degranulation of large, dense platelets. Thus platelet size and density are independently determined and the increased size of platelets in immune thrombocytopenia may be the result of abnormalities in their production.     

Sustained thrombocytopenia in mice: serial studies of megakaryocytes and platelets

We studied thrombopoiesis in mice after the experimental induction of sustained, immune thrombocytopenia with platelet antiserum (PAS). Utilizing light and electron microscopy and a digital image analyzer to determine platelet sectional areas, we examined platelets and megakaryocytes (MK) after 120 h of sustained, severe thrombocytopenia (120CT) and during recovery from thrombocytopenia at 48 h (48R), 72 h (72R), and 120 h (120R) after cessation of administration of PAS. Mean platelet volume (MPV), determined by electrical impedance, also was measured at each time point. Platelets at 120CT (platelet count less than 50,000/microliter), 48R (platelet count 100-200,000/microliter), and 72R (platelet count approximately 1 x 10(6)/microliter) were significantly larger in sectional area than control platelets and contained increased profiles of endoplasmic reticulum and Golgi cisternae, a lower concentration of surface-connected canalicular system, and occasional membrane complexes. The largest median platelet sectional area was detected at 48R and was the largest median value observed in response to either chronic or acute thrombocytopenia. At 120R, most platelets were normal in size and cytoplasmic appearance, although some large cells remained present in the circulation. MPV paralleled the morphometric changes in platelet sectional area. MK were increased in number at 120CT, 48R, 72R, and 120R. In addition, at least half of the MK examined at 48R contained small areas of cytoplasm, devoid of organelles, that were interspersed between larger areas of organelle-filled, undemarcated cytoplasm. The modal bone marrow megakaryocyte ploidy class, determined using two-color fluorescence-activated flow cytometry, shifted from 16N to 32N in response to sustained thrombocytopenia. In contrast, during recovery and development of rebound thrombocytosis, the relative frequency of 8N megakaryocytes was significantly increased. Because there was no consistent correlation between megakaryocyte cytoplasmic characteristics and platelet morphology, these data support the hypothesis that platelet formation is not determined by compartmentalization of MK cytoplasm into platelet areas as MK mature in the bone marrow, but involves a rearrangement of MK cytoplasm immediately prior to platelet release.     

Autoimmune thrombocytopenia in sarcoidosis

Severe thrombocytopenia and splenomegaly developed in a young man with sarcoidosis. Platelet-associated immunoglobulin (IgG) was strongly positive, and platelet survival studies revealed a half-life of five and a half hours. Treatment with prednisone and vincristine led to a rise in the platelet count to 100,000/mm3 after two months with no change in the splenomegaly. Five months later, when the platelet count was normal, the level of platelet-associated IgG had fallen to normal. Repeated platelet survival studies showed an initial half-life of three hours with a second half-life of two days, associated with accumulation in the spleen. Although there was evidence for splenic sequestration of platelets, the dominant mechanism of thrombocytopenia appeared to be antibody-mediated destruction, analogous to that seen in idiopathic autoimmune thrombocytopenic purpura and responsive to immunosuppressive therapy.     

Adenovirus-induced thrombocytopenia: the role of von Willebrand factor and P-selectin in mediating accelerated platelet clearance

Thrombocytopenia has been consistently reported following the administration of adenoviral gene transfer vectors. The mechanism underlying this phenomenon is currently unknown. In this study, we have assessed the influence of von Willebrand Factor (VWF) and P-selectin on the clearance of platelets following adenovirus administration. In mice, thrombocytopenia occurs between 5 and 24 hours after adenovirus delivery. The virus activates platelets and induces platelet-leukocyte aggregate formation. There is an associated increase in platelet and leukocyte-derived microparticles. Adenovirus-induced endothelial cell activation was shown by VCAM-1 expression on virus-treated, cultured endothelial cells and by the release of ultra-large molecular weight multimers of VWF within 1 to 2 hours of virus administration with an accompanying elevation of endothelial microparticles. In contrast, VWF knockout (KO) mice did not show significant thrombocytopenia after adenovirus administration. We have also shown that adenovirus interferes with adhesion of platelets to a fibronectin-coated surface and flow cytometry revealed the presence of the Coxsackie adenovirus receptor on the platelet surface. We conclude that VWF and P-selectin are critically involved in a complex platelet-leukocyte-endothelial interplay, resulting in platelet activation and accelerated platelet clearance following adenovirus administration.     

Successful corticosteroid treatment of thrombocytopenia in a pregnant woman with myelodysplastic syndrome (refractory anemia)

A 31-year-old pregnant woman was referred to our hospital due to anemia and thrombocytopenia, and was diagnosed as having myelodysplastic syndrome (refractory anemia) with autoimmune thrombocytopenia. Administration of high dose methylprednisolone and gamma-globulin did not raise her platelet count, and she subsequently delivered a healthy baby after the transfusion of a large amount of platelets. Although the anemia spontaneously improved after delivery, the platelet count remained unchanged. Prednisolone was thus administered a second time, which did finally increase the platelet count. This is the first reported case of a pregnant woman with myelodysplastic syndrome in whom corticosteroid administration was effective for thrombocytopenia.     

Platelet Volume: Density Relationships in Normal and Acutely Bled Dogs

The relationship between platelet volume and platelet density was studied in normal dogs and in dogs subjected to acute blood loss.
Frequency distribution histograms of platelet densities were obtained by centrifuging diluted whole blood over a series of 13 silicone solutions varying in specific gravity from 1.007 to 1.065 and determining the percentage of platelets that remained in the supernatants above the silicone after centrifugation for 2 min at 9500 g. Frequency distribution histograms of platelet volumes were obtained using the Model B Coulter Counter with the Coulter Model H particle size analyser.
Platelets of normal dogs have a skewed distribution of densities. The mean density is approximately 1.020 but the plateau region on the cumulative distribution of platelet densities is not reached until specific gravity 1.045-1.050. Mean platelet volume increases as density increases.
When platelet production was increased following acute blood loss, both mean density and mean volume increased abruptly, but the change in density histograms was more prolonged as well as more striking than change in mean density. Results of serial observations suggest that the large dense platelets produced following acute blood loss represent an aberrant population and that these platelets do not decrease in volume as they age.     

Relationship between size and thiazole orange fluorescence of platelets in patients undergoing high-dose chemotherapy.

The reticulated platelet count relies upon the assumption that newly formed platelets contain a residual amount of RNA which selectively binds the dye thiazole orange (TO) and greatly enhances its fluorescence signal. It has, however, recently been shown that almost half of the platelet TO-signal is derived from the labelling of dense-granule nucleotides. It is therefore possible that the higher TO fluorescence of young platelets partially derives from the higher granule content due to their larger volume. To investigate the relationship between platelet size and TO fluorescence we studied 13 patients with high-risk breast cancer undergoing high-dose chemotherapy. Mean platelet volume, platelet distribution width, platelet-large cell ratio, membrane content of glycoprotein Ib and IIb-IIIa and platelet aggregation were significantly greater during resolution than during development of thrombocytopenia, suggesting a prevalence of young and old platelets respectively. Mean TO fluorescence per cell was higher in the platelet population enriched in young cells than in that enriched in old cells, but this difference was no longer observed when the ratio TO signal/platelet size was examined. Moreover, RNase treatment and platelet degranulation reduced TO fluorescence to a similar extent in platelet populations enriched in young or old cells. Therefore our data suggest that the higher TO signal of young platelets is derived, to a significant extent, from their larger volume and granule content.     

Platelet Heterogeneity

Human platelets were separated into 2 density populations by repeated centrifugations of platelet-rich plasma at increasing gravitational force. The heaviest platelet fraction was rich in larger platelets. The lightest platelet fraction was rich in smaller platelets. In both fractions and in the platelet button, lipid peroxidation (malonaldehyde-MDA-production after addition of thrombin) was measured at basal condition, on the 1st, 3rd, 5th, 7th and 9th day after aspirin ingestion. At basal conditions and after ingestion of aspirin, MDA production was higher in the heavy-large platelets than in light-small ones, but a parallel increase of MDA production was observed in the light and in the heavy population and in the platelet button. The data are not compatible with the hypothesis that platelet density and size are age-related. Aspirin inhibits platelet lipid peroxidation by permanently acetylating their cyclooxygenase and if the heaviest platelets were the young ones, lipid peroxidation should reappear sooner in them.     

An acquired Bernard-Soulier-like platelet defect associated with juvenile myelodysplastic syndrome

Bernard-Soulier syndrome is an inherited bleeding abnormality characterized by thrombocytopenia with large platelets and deficiency of the platelet membrane glycoprotein (GP) Ib-IX complex. We have identified a young female with an acquired Bernard-Soulier-like platelet defect and a coexisting primary myelodysplastic disorder. Abnormal bruising had developed at age 5. A normal platelet count with some giant platelets was noted at age 7. At age 9 she developed a large haematoma following surgery. Laboratory investigation revealed thrombocytopenia and large platelets. Platelet membrane glycoprotein analysis showed a marked deficiency of the components of the GP Ib-IX complex (approximately equal to 25% of normal). Flow cytometry revealed two populations of platelets: a predominant population of large platelets lacking the GP Ib-IX complex and a minor population of normal-sized platelets with normal GP Ib-IX expression. The patient developed progressive anaemia, more severe thrombocytopenia and neutropenia, and circulating blast cells were seen. A bone marrow showed gross hypercellularity with marked dysplasia of all three lineages and increased blasts. Marrow cytogenetic studies showed the presence of monosomy 7 in all metaphases, with an additional trisomy 21 in 10%. Peripheral blood cells were normal 46XX. The above data are consistent with an acquired myelodysplastic syndrome associated with a Bernard-Soulier-like platelet defect.     

Platelet heterogeneity. Relationship between buoyant density, size, lipid peroxidation and platelet age

Human platelets were separated into 2 density populations by repeated centrifugations of platelet-rich plasma at increasing gravitational force. The heaviest platelet fraction was rich in larger platelets. The lightest platelet fraction was rich in smaller platelets. In both fractions and in the platelet button, lipid peroxidation (malonaldehyde-MDA-production after addition of thrombin) was measured at basal condition, on the 1st, 3rd, 5th, 7th and 9th day after aspirin ingestion. At basal conditions and after ingestion of aspirin, MDA production was higher in the heavy-large platelets than in light-small ones, but a parallel increase of MDA production was observed in the light and in the heavy population and in the platelet button. The data are not compatible with the hypothesis that platelet density and size are age-related. Aspirin inhibits platelet lipid peroxidation by permanently acetylating their cyclooxygenase and if the heaviest platelets were the young ones, lipid peroxidation should reappear sooner in them.     

A case of malignant lymphoma producing autoantibody against platelet glycoprotein Ib

A 61-year-old woman was referred to our hospital for refractory thrombocytopenia (3 x 10(3)/microliter) and massive melena. Bone marrow aspiration revealed normal cellularity and increased megakaryocytes (250/microliter). An abdominal computerized axial tomography scan showed isodensity masses on both adrenal glands. 67 Ga-scintigraphy exhibited strong uptake into the bilateral adrenal tumor and mediastinal region. IgM-type antibody against platelet glycoprotein Ib (GpIb) was detected in the patient's serum. A needle biopsy of the right adrenal tumor was performed, and histology was non-Hodgkin's lymphoma (NHL), diffuse large B-cell type. Following the diagnosis of autoimmune thrombocytopenia associated with lymphoma, administration of corticosteroid (predonisolone 60 mg/day) and high-dose intravenous globulin (15 g/day x 4 days) was carried out, but neither was effective in normalizing the thrombocytopenia. Immunosuppressive therapy (cyclophosphamide 500 mg and 1 mg of vincristine) markedly restored the platelet counts to 7.2 x 10(4)/microliter and ceased the melena; furthermore, the size of adrenal tumors decreased by more than 60% after therapy. We cultured the lymphoma cells drawn by needle biopsy with IL-6 in vitro and found that the lymphoma cells produced IgM-type antiplatelet antibodies against platelet GpIb in the culture supernatant. Thus this is a rare case of NHL in which the production of antiplatelet antibody from lymphoma cells was confirmed in vitro.     

Thrombocytopenia in dogs induced by granulocyte-macrophage colony- stimulating factor: increased destruction of circulating platelets

Administration of recombinant canine granulocyte-macrophage colony- stimulating factor (rcGM-CSF) to normal dogs in previous studies induced an increase in peripheral blood neutrophils and a dose- dependent decrease in platelet counts. In six dogs that received the highest tested dose of rcGM-CSF (50 micrograms/kg/d) for a minimum of 12 days, the mean nadir of the platelet count was 46,000/microL (range, 4,000 to 91,000/microL) on day 9 +/- 1.1 after starting therapy, compared with a mean baseline platelet count of 398,000/microL (range, 240,000 to 555,000/microL). In three dogs, survival of autologous 111In- labeled platelets was reduced from a mean of 4.9 days to 1.3 days during the administration of rcGM-CSF. Biodistribution studies with gamma camera imaging indicated that there was an increase in mean hepatic uptake during the administration of rcGM-CSF, from 15% to 44% of the total injected 111In-labeled platelets at 2 hours, whereas splenic uptake was not significantly changed. In contrast, in two evaluable dogs who were recipients of 111In-labeled platelets from matched allogeneic donors receiving rcGM-CSF, platelet survival was not reduced and no increased hepatic uptake was noted. A third dog became alloimmunized to the matched donor platelets and was not evaluable. Immunohistologic studies of liver and spleen were performed with monoclonal antibodies specific for canine gpIIb/IIIa and P-selectin in dogs treated with rcGM-CSF and compared with untreated controls. On treatment, a marked reduction of platelets in the red pulp of the spleen was evident, and in general, the presence of platelet antigen in the liver was unchanged. Therefore, platelets were not being sequestered, but destroyed in the liver and spleen. The platelet antigens, P-selectin and gpIIb/IIIa, were identified in association with Kupffer cells in the liver, but no difference in the number of distribution of these Kupffer cells was found between controls and rcGM- CSF-treated dogs. In the spleen during rcGM-CSF treatment, most platelet antigens were associated with large mononuclear cells in the marginal zone. During administration of rcGM-CSF, CD1c and CD11c expression was increased on Kupffer cells. Platelet P-selectin expression and binding of leukocytes to circulating platelets were unchanged from baseline studies with rcGM-CSF treatment. In conclusion, during the administration of rcGM-CSF to dogs, a local process in the liver and spleen is induced resulting in thrombocytopenia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inverse relationship between platelet density and reactivity alterations at coronary angiography.

This work investigates relationships between platelet density and reactivity. 21 individuals subject to coronary angiography were studied. Peak platelet density was analyzed using a newly developed electronic device. The apparatus measures light transmission through test tubes containing density-separated platelets, thus allowing an estimation of the platelet distribution in the gradient. A flow cytometry technique was used for determining platelet reactivity after stimulating with ADP. Platelet counts, mean platelet volumes, peak platelet density and platelet reactivity were determined immediately before (day 1) and 24 h after cardiac catheterization (day 2). For all parameters changes during the day of angiography were compared with platelet density alterations. The subjects were divided into two groups according to density changes at angiography. Group 1 individuals showed density alterations (i.e. day 2 - day 1 value) > or = -8 x 10(-5) kg/l. In contrast, group 2 subjects either displayed density changes < -8 x 10(-5) kg/l or grossly disturbed platelet density patterns on day 2. Before angiography both groups had similar platelet counts and volumes. Then platelet reactivity when stimulating with ADP did not differ significantly between the two groups. After angiography, the number of fibrinogen-positive cells when stimulating with ADP rose by 6 +/- 8% for group 2 patients. The corresponding figure for group 1 was -1 +/- 6%. The difference was significant (p = 0.01). No such relationships were found when comparing density alterations and changes of platelet counts and volumes. We conclude that in this study platelet density alterations at coronary angiography are inversely related to variations of platelet reactivity.     

Platelet size and age determine platelet function independently

This study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se- methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms.