Direct flow cytometric measurement of nucleic acid content in individual platelets is possible using the fluorescent dye Thiazole Orange (Becton-Dickinson, San Jose, CA). When applied to studies of thrombocytopenic patients, platelets with elevated nucleic acid content ("reticulated platelets") can be identified and quantitated. Labeling of these platelets is saturable and is abolished by treatment with RNAse. It has been suggested that, similar to the erythrocyte reticulocyte response to anemia, the number of these platelets appearing in the circulation may provide an estimate of the rate of thrombopoiesis. The authors studied 229 thrombocytopenic patients, measuring both reticulated platelets and platelet-associated immunoglobulin. The results show that for the subset of patients with normal levels of platelet-associated immunoglobulin, the average absolute number of reticulated platelets is independent of platelet count and remains in the normal range. For those with elevated levels of platelet-associated immunoglobulin, the absolute number of reticulated platelets increases in patients who are moderately thrombocytopenic (60 to 100 x 10(9)/L) but decreases to normal or subnormal levels as thrombocytopenia worsens. The latter finding has been duplicated in studies of mice made thrombocytopenic by injection of anti-platelet antiserum. These results are consistent with the hypothesis that reticulated platelets are subject to peripheral destruction at the same rate as mature platelets, and that in the severely thrombocytopenic patient their level may decrease despite an appropriate marrow thrombopoietic response.

Platelets are known to become activated during storage, but it is unclear whether such activation affects recovery or survival after platelet concentrate (PC) transfusion. With the use of flow cytometry to determine the percentage of platelets expressing the alpha-granule membrane protein 140 (GMP-140), a known adhesive ligand appearing on the platelet surface after activation, several studies were conducted. These investigations evaluated 1) the occurrence of significant platelet activation over time in PCs (n = 46) stored under standard blood bank conditions; 2) the correlation between platelet activation and platelet recovery in normal subjects after PC storage (n = 12), as assessed by the recovery of Indium-labeled platelets; and 3) the recovery of activated and unactivated platelets in thrombocytopenic cancer patients transfused with standard PCs (n = 11). It was determined 1) that an increasing duration of storage of PC was associated with increasing platelet activation as measured by the percentage of platelets expressing GMP-140, progressing from a mean of 4 +/- 2 percent (SD) on the day of collection to a mean of 25 +/- 8 percent by 5 days of storage: 2) that, in normal subjects, posttransfusion recovery of autologous platelets stored for 2 to 4 days and then labeled with In111 was inversely correlated with the percentage of activated platelets in the transfused PC (r = -0.55, p = 0.05); and 3) that, when thrombocytopenic patients were transfused with standard PCs, the recovery of the activated platelets in the transfused PCs averaged only 38 +/- 15 percent of the number predicted by the absolute platelet increment.(ABSTRACT TRUNCATED AT 250 WORDS)

Immune interferon (IFN-y) increases the surface expression of HLA-A,B antigens and induces the surface expression of HLA-DR antigens on vascular endothelial cells and dermal fibroblasts. Here we report that IFN-y induces parallel expression of two other class H major histocompatibility complex (MHC) antigens, SB and DC. Maximal surface expression of all three antigens is reached in 4-6 days, and HLA-DR and -SB are induced to a higher level of expression than HLA-DC. For all three class II antigens, induction is marked by the de novo appearance of detectable transcripts of class II heavy and light chains and of the non-MHC-encoded invariant chain, suggestive of the transcription of multiple previously silent genes. Class I message levels and antigen expression are also increased by IFN-y at similar rates but from initial levels that are 50% of maximal. After removal of IFN-y, class II antigen expression persists for at least 4 days, while mRNA levels decrease rapidly. The parallel induction and persistence of the several class II MHC antigens may be important in conferring immune accessory function on vascular and stromal cells. 8).To date, three sets of human class II antigens (HLA-DR, -DC, and -SB) have been identified. All three sets of antigens have been studied at the protein and nucleic acid level; they consist of similar heterodimers with heavy chains of --34 kDa, and light chains of "-29 kDa (1). Biosynthetic intermediates, but not mature surface-expressed class II antigens, appear associated with a non-MHC non-polymorphic "invariant" chain of ==31 kDa (9). In human, but not in murine cells, class II MHC loci may be independently expressed (10-12). Furthermore, differential expression (e.g., DR', DC-) may be correlated with limitations on the ability of the class II antigen-bearing cell to interact with responding T cells (10-12). To understand more fully the potential of endothelial and stromal cells to interact with T cells, the effects of IFN-y on these cells have been studied in greater detail, and it has been found that: (i) recombinant human IFN-y induces the de novo appearance of transcripts of multiple class II antigen genes, including the a and f3 genes of HLA-DR, -DC, and -SB, as well as transcripts of the invariant (-y) chain, with subsequent surface expression of all three class II antigens; and (il) after removal of IFN, the class II-specific mRNAs disappear relatively rapidly, while the antigens remained expressed on the surface at relatively constant levels for several days. MATERIALS AND METHODSHUVE cells were isolated and grown in primary culture as described (13). In some experiments, HUVE cells were passaged (5-10 subcultures) in medium containing 100 Ag of endothelial cell growth factor per ml (a generous gift of Thomas Maciag, Meloy Laboratories, Springfield, VA) and porcine heparin (Sigma) on 0.1% gelatin-coated (Difco) dishes (14). HDF (a generous gift of James Rheinwald, Dana-Farber Cancer Institute, Boston; see ref. 15) strains were cultured as described (8). HUVE and HDF...

Abstract-Endothelial nitric oxide synthase (eNOS) has been identified in human platelets. Although platelet-derived nitric oxide (NO) has been shown to inhibit platelet recruitment in vitro, its role in the regulation of the hemostatic response in vivo has not been characterized. To define the role of platelet-derived NO in vivo, we studied mice that lacked a functional eNOS gene (NOSIII). Surface P-selectin expression in platelets from eNOS-deficient mice was not significantly altered; however, bleeding times were markedly decreased in eNOS-deficient versus wild-type mice (77.2Ϯ3 versus 133.4Ϯ3 seconds, PϽ0.00005). To determine the contribution of endothelium-versus platelet-derived NO to the bleeding time, isolated platelets from either eNOS-deficient or wild-type mice were transfused into a thrombocytopenic eNOS-deficient mouse and the bleeding time was measured. The bleeding times in mice transfused with eNOS-deficient platelets were significantly decreased compared with mice transfused with wild-type platelets (⌬bleeding time, Ϫ24.6Ϯ9.1 and Ϫ3.4Ϯ5.3 seconds, respectively; PϽ0.04). Platelet recruitment was studied by measuring serotonin release from a second recruitable population of platelets that were added to stimulated platelets at the peak of NO production. There was 40.3Ϯ3.7% and 52.0Ϯ2.1% serotonin release for platelets added to wild-type or eNOS-deficient platelets, respectively (PϽ0.05). In summary, mice that lacked eNOS had markedly decreased bleeding times even after endothelial NO production was controlled. These data suggest that the lack of platelet-derived NO alters in vivo hemostatic response by increasing platelet recruitment. Thus, these data support a role for platelet-derived NO production in the regulation of hemostasis. (Circ Res. 1999;84:1416-1421.)Key Words: selectin Ⅲ mice Ⅲ platelet Ⅲ nitric oxide synthase N ormal hemostatic balance is maintained by tight regulation of platelet activation and recruitment. Adhesion of platelets to the endothelium is prevented by several mechanisms, including endothelial cell production of nitric oxide (NO). 1,2 NO inhibits platelet adhesion and aggregation 3,4 and prevents thrombosis. 5 Exogenous NO inhibits the normal activation-dependent increase in the expression of platelet surface glycoproteins, including P-selectin and the integrin glycoprotein IIb/IIIa complex. 6 In addition, we have shown that a functional decrease in exogenous NO can lead to a clinical thrombotic disorder. 7,8 In addition to its presence in endothelial cells, constitutive NO synthase (NOS) has been identified in human platelets and megakaryocytic cells. 9 -12 Consistent with these observations, studies report NO release from aggregating platelets. [13][14][15] Platelet aggregation is enhanced by incubation with inhibitors of NOS and inhibited by incubation with the NOS substrate L-arginine. 16 Although platelet-derived NO appears to inhibit the primary aggregation response only modestly, we have recently shown that NO release from activated human platelets markedly inhibits ...