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Administration of 5-fluorouracil (5-FU) to mice results in a marked increase in the level of circulating platelets in 10 days. Mice lacking Mpl, the receptor for thrombopoietin (TPO), are thrombocytopenic. To gain insight into the mechanism by which 5-FU produces such a substantial stimulation of platelet production, this study investigated whether 5-FU (150 mg/kg) produced thrombocytosis in c-mpl−/− mice, thus establishing whether TPO was required for this response. A 5- to 6-fold increase in platelet levels in c-mpl−/− mice (to approximately 1000 × 109/L) was observed on days 20 and 25 after 5-FU injection. Thus, at the peak of the response, c-mpl−/− mice had platelet levels comparable to those in normal mice. Administration of 5-FU also produced thrombocytosis in previously splenectomized c-mpl−/− mice. Comparison of the platelet response to 5-FU in young (6-12 weeks) and old (33-46 weeks) c-mpl−/− mice found that older mice produced a much more marked response than younger mice, with a mean maximum platelet level of approximately 1700 × 109/L. To determine whether this increase in circulating platelets was preceded by an increase in hematopoietic progenitors, serial cultures of bone marrow and spleen were evaluated. A considerable increase in all colony types studied was observed on days 15 and 20 in spleens of c-mpl−/− mice, but no similar elevations were detected in bone marrow. These results indicate that c-mpl−/− mice can achieve a normal level of platelets after 5-FU injection, by means of a TPO-independent mechanism, and that they respond to 5-FU myelosuppression by producing large numbers of megakaryocytic, myeloid, and erythroid progenitors.

Administration of 5-fluorouracil (5-FU) to mice results in a marked increase in the level of circulating platelets in 10 days. Mice lacking Mpl, the receptor for thrombopoietin (TPO), are thrombocytopenic. To gain insight into the mechanism by which 5-FU produces such a substantial stimulation of platelet production, this study investigated whether 5-FU (150 mg/kg) produced thrombocytosis in c-mpl−/−mice, thus establishing whether TPO was required for this response. A 5- to 6-fold increase in platelet levels in c-mpl−/−mice (to approximately 1000 × 109/L) was observed on days 20 and 25 after 5-FU injection. Thus, at the peak of the response, c-mpl−/−mice had platelet levels comparable to those in normal mice. Administration of 5-FU also produced thrombocytosis in previously splenectomized c-mpl−/−mice. Comparison of the platelet response to 5-FU in young (6-12 weeks) and old (33-46 weeks) c-mpl−/−mice found that older mice produced a much more marked response than younger mice, with a mean maximum platelet level of approximately 1700 × 109/L. To determine whether this increase in circulating platelets was preceded by an increase in hematopoietic progenitors, serial cultures of bone marrow and spleen were evaluated. A considerable increase in all colony types studied was observed on days 15 and 20 in spleens of c-mpl−/−mice, but no similar elevations were detected in bone marrow. These results indicate that c-mpl−/−mice can achieve a normal level of platelets after 5-FU injection, by means of a TPO-independent mechanism, and that they respond to 5-FU myelosuppression by producing large numbers of megakaryocytic, myeloid, and erythroid progenitors.

It is known that at 37°C plasmin may have two opposite effects on platelets: at high concentrations (>1.5 caseinolytic units [CU]/mL), plasmin activates platelets; at lower concentrations (0.1 to 1.0 CU/mL) it inhibits platelet activation induced by thrombin, collagen, or calcium ionophore A23187. In this study, we report that when lowering the incubation temperature to 22°C, plasmin at low concentrations (0.1 to 0.5 CU/mL) fully activated platelets. When platelets were treated with 0.2 CU/mL of plasmin, lowering the incubation temperature from 37°C to 22°C resulted in an increase in the expression of fibrinogen receptors, in platelet release and aggregation. Thromboxane A2was not generated by plasmin treatment at either temperature. Ultrastructural studies showed that platelets responded to low-dose plasmin at 37°C by forming pseudopods, centralizing granules without fibrinogen release, whereas at 22°C the same dose of plasmin caused platelet degranulation with the appearance of α-granule fibrinogen within the lumen of the surface connected canalicular system. In addition, at 22°C plasmin at doses insufficient to induce platelet aggregation potentiated platelet response to thrombin. Thus, we suggest that plasmin may initiate both activating and inhibitory processes within platelets and that the change of temperature could influence this balance. These results may be of clinical relevance, because the fibrinolytic system was found activated during cardiopulmonary bypass in which the temperature of patient's blood circulation was reduced. This temperature-dependent behavior is also an interesting model for a further study on platelet response to serine proteinases.

Platelet factor 4 (PF-4) inhibits angiogenesis in vitro and in vivo. The mechanism of inhibition is poorly understood. We have investigated the mechanism of inhibition by examining the interaction of PF-4 and the fibroblast growth factor-2 (FGF-2)/fibroblast growth factor receptor (FGFR) system. PF-4 inhibited the binding of FGF-2 to high-affinity and low-affinity binding sites in murine microvascular endothelial cells (LEII cells) and proliferation. Maximum inhibition of binding to endothelial FGF receptors was observed at PF-4 concentrations between 5 and 10 µg/mL (half maximum inhibition at 0.6 µg/mL), and proliferation was completely inhibited at 2 µg/mL. At this concentration, PF-4 reduced internalization of125I–FGF-2 by threefold and delayed degradation. To gain insight into the mechanism of inhibition, we have analyzed the interaction of PF-4 with FGF-2/FGFR by using mutant heparan sulfate–deficient Chinese hamster ovary (CHO) cells transfected with the FGFR-1 cDNA (CHOm–FGFR-1) and by examining the direct interaction with FGF-2. In the absence of heparin, PF-4 inhibited binding of 125I–FGF-2 to CHOm–FGFR-1 cells in a concentration-dependent manner, although not completely. In the presence of heparin, PF-4 abolished totally the stimulatory effect of heparin. Furthermore, PF-4 complexed to FGF-2 and inhibited endogenous or heparin-induced FGF-2 dimerization. These results indicate that PF-4 interacts with FGF-2 by complex formation, inhibiting FGF-2 dimerization, binding to FGF receptors, and internalization. This mechanism most likely contributes to the antiangiogenic properties of PF-4.

Washed human platelets were labeled with I by the lactoperoxidase-catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated Ia, Ib, IIa, IIb. IIIa, and IIIb. Glycoproteins IIb and IIIa were shown to be contained in a single immunoprecipitate, while glycoproteins Ia and IIa were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann’s thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins IIb and IIIa. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein Ib.

To examine the control of porcine von Willebrand factor (vWF) biosynthesis we cloned human vWF complementary DNA (cDNA) and investigated the expression of the vWF gene in lungs from normal pigs and pigs with severe von Willebrands disease (vWD). Recombinant clones spanning approximately 90% of human vWF cDNA were identified in a X gt10 human lung cDNA library by screening with oligonucleotides. One clone spanning nucleotides 960 to 3,240 of human vWF cDNA was used to investigate the steady-state levels of vWF mRNA in lungs from normal pigs and from pigs phenotypically determined to be homozygous for vWD. This clone hybridized with genomic DNA from pig leukocytes when Southern blots were processed under very stringent conditions; therefore, human cDNA clones were considered valid probes to detect porcine mRNA. Northern blot analysis of total RNA from normal pig lung and human umbilical vein endothelial cells identified the vWF mRNA as a molecular species of approximately 9.0 kilobases (kb). A very faint to undetectable band at 9.0 kb in total RNA from lungs of vWD pigs suggested a decreased rate of transcription of the vWF gene. Sucrose density gradient centrifugation of RNA from the vWD pigs confirmed by Northern analysis that the high-molecular weight fractions contained vWF mRNA and at the same size as normal pig mRNA. Dot blot hybridization analysis of vWF and actin mRNA processed under stringent conditions demonstrated that the relative ratio of vWF mRNA to actin mRNA in the vWD pigs varied from 21 % to 41 % of the ratio observed in normal pigs. Because the amount of vWF mRNA is not correlated to the amount of vWF activity or antigen in plasma of vWD pigs we conclude that posttran-scriptional events are also probably involved in abnormal expression of vW7 in these animals.

In subagglutinating amounts, an IgG antibody isolated from the plasma of a polytransfused thrombasthenic patient (L) inhibited ADP-, epinephrine-, collagen-, and thrombin-induced aggregation of normal human platelets. The inhibition of ADP-induced aggregation was strongly diminished following the prior incubation of the antibody with control human platelet stroma but not with the stroma prepared from the platelets of two different thrombasthenic patients. The IgG(L) did not affect the binding of 14C-ADP to control human platelet membranes and did not inhibit the ADP-induced shape change.

The autosomal recessive transmission of Glanzmann's thrombasthenia (GT) and Bernard-Soulier syndrome (BSS), together with requests of families who already had children with these diseases, prompted us to investigate the feasibility of their antenatal diagnosis. The preliminary step leading to the early detection of GT or BSS was to characterize, in the normal human fetus, the platelet antigens and glycoproteins (GPs) and to define their normal amounts on the membrane surface. Blood samples from 32 fetuses between 18 to 26 weeks of gestation were collected by direct puncture of the umbilical vein using an ultrasound-guided needle. Polyclonal antibodies from human origin directed against PLA1, Lekaantigens, and the GPllb llla complex (IgGL), or murine monoclonal antibodies specific for GPlb (AN51, 6D1), GPllla (AP-3), or GPllb llla (AP-2) were studied using platelet suspension immunofluorescence tests. The binding of each antibody was quantified using a cytofluorograph (Ortho 50H). PLA1 and Lekaantigens were expressed in normal amounts on fetal platelets as early as 16 weeks of intrauterine life. The GPllb llla complex quantified by polyclonal or monoclonal antibodies was in the same range in fetuses (lgGL = 427 ± 23 AUF, AP-2 = 459.5 ± 8.5; AP-3 = 536 ± 14) and in adults (IgGL = 420 ± 30; AP-2 = 498 ± 11; AP-3 = 515 ± 13). The platelet binding of antibodies that recognized GPlb was higher in fetuses (AN51 = 491.5 ± 14; 6D1 = 479 ± 15) than in adults (AN51 = 426.5 ± 9; 6D1 = 449 ± 8.7). These results suggest that immunological techniques can be applied as early as 18 weeks of gestation for the antenatal diagnosis of GT and BSS.

The gray platelet syndrome is a rare inherited platelet disorder characterized by the absence of α-granules as observed by electron microscopy. Analysis of the glycoprotein composition of the platelets of 2 such patients by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed decreased or absent staining for carbohydrate of several high molecular weight glycoproteins. The major periodic acid Schiff (PAS) staining membrane glycoproteins were normally detected and were normally labeled with 125I during lactoperoxidase-catalyzed iodination. Analysis of the protein composition of gray platelets by single or two-dimensional SDS-PAGE followed by Coomassie blue staining revealed an apparent absence of GP Ig (thrombo-spondin), markedly reduced platelet fibrinogen and albumin concentrations, and severely reduced levels of 2 low molecular weight polypeptides exhibiting identical rates of migration on SDS-PAGE as platelet factor 4 and β-thromboglobulin. SDS-PAGE profiles similar to those of the gray platelets were observed with normal human platelets that had undergone the release reaction induced by thrombin. Analysis of gray platelet proteins by crossed Immunoelectrophoresis using a rabbit anti-human platelet antibody preparation and rocket immunoelectrophoresis using monospecific antisera confirmed the above findings and showed additional severe deficiencies of factor VIIIR:Ag and cold-insoluble globulin. In contrast, factor XIII (subunit A), a cytoplasmic protein, was normally detected. Our studies provide further evidence that circulating gray platelets specifically lack, or have markedly decreased concentrations of, the α-granule proteins.

Short peptides isolated from fibrinogen and K-casein have been shown to inhibit platelet aggregation and fibrinogen binding to stimulated platelets. We studied the effects of synthetic peptides occurring in milk proteins (bovine K-casein, KNQDK, and human lactotransferrin, KRDS) and in fibrinogen (RGDS and L10) on subsequent binding of monoclonal antibodies (MoAb) against the glycoprotein (GP) Ilb-IIIa complex (AP2and P2) on adenosine diphosphate (ADP)-stimulated and unstimulated human platelets and megakaryocytes (MKs) by using an immunoperoxidase method to visualize antibody binding. Only KRDS (900 μmol/L) inhibited the binding of AP2and P2on ADP (5 μmol/L)-stimulated platelets, but not on unstimulated platelets. However, the binding of P2was considerably more inhibited than that of AP2as judged by immunoperoxidase intensity. Radiolabeled AP2binding was inhibited by 30% with KRDS on ADP-stimulated platelets as compared with platelets incubated in the absence of ADP. KRDS did not inhibit the binding of MoAbs against GP Ilia (SZ 21), GP lib (SZ 22), and GP lb (SZ 2) on ADP-stimulated human platelets. Inhibition of P2binding by KRDS was also observed in a section of MKs isolated from human bone marrow and stimulated by 15 or 20 Mm ADP. A lower concentration of ADP (5 or 10 Mmol/L) failed to produce any inhibition of binding. This indicates that MKs may not be equally responsive to agonists as platelets. Moreover, P2binding inhibition was observed in a larger (P<.001) percentage of mature MKs (29%) as compared with younger, maturing MKs (11%). The observations suggested that a functional ability possessed by platelets, namely, agonistinduced exposure of the site of interaction of KRDS, may occur at a late stage of MK development.

An IgG antibody isolated from the serum of a patient with the Bernard-Soulier syndrome induced platelet agglutination in the platelet-rich plasma of 50 normal subjects regardless of their ABO, K0a, K0b, HLA, or PIA1types. This antibody was nonreactive with platelets from three other Bernard-Soulier syndrome patients. Indirect immunoprecipitation tests using this serum (or purified IgG) and soluble membrane antigens labeled with 125I that had been extracted from normal platelets by the nonionic detergent Nonidet P-40 gave a single radioactive peak at 150,000 MW in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These findings strongly suggest that the antigenic determinant reacting with this antibody is absent from platelets of Bernard-Soulier syndrome patients and that the deficient molecule is of 150,000 MW. The role of this molecule in subendothelial adhesion and macromolecular-mediated aggregation is discussed.

Thrombin bound to a fibrin clot remains active and poorly accessible to heparin-AT III complex. During fibrinolysis, thrombin is released as thrombin-FDP complex and is inactivated by heparin-AT III. However, as successive fibrin layers are removed, inaccessible molecules of thrombin are exposed at the surface of the residual clot, possibly contributing to the occurrence during thrombolytic therapy of coagulation that is poorly controlled by heparin. We have investigated the accessibility of fibrin-bound thrombin to hirudin. The results clearly show that two recombinant hirudin variants neutralize thrombin both in solution and fibrin bound. Furthermore, we have found that in in vitro models, hirudin present in the surrounding medium of a clot under lysis is more efficient than heparin in preventing the activation of coagulation. This observation suggests that hirudin may be effective in the prevention of the rethrombotic process frequently encountered during thrombolytic therapy.

The electron microscopic localization of von Willebrand factor (vWF) was studied in platelets from normal and von Willebrand disease (vWD) pigs. In normal pig platelets, immunolabeling for vWF was far more intense and extensive than in human platelets and was either localized at one pole of the a-granule or all along its periphery or long axis. As in human platelets, this immunolabeling coincided with the presence of tubules about 200nm in diameter. These structures were more numerous than in human platelets, with up to 30 tubules per a-granule. They were easily identified either in transverse sections, usually grouped in a less electron-dense part of the matrix at one pole of the a-granule, or in longitudinal sections parallel to the long axis of the elongated granules, or coiled around the α-granule core. They closely resemble those structures found in Weibel-Palade bodies. In platelets from pigs with severe vWD, these structures were absent, as was the immunolabeling for vWF; however, cytoplasmic microtu-bules were normally present in these platelets. Thus, the granule-associated tubules can be distinguished from the microtubules, which are larger in diameter (250nm), are present in both normal and vWD platelets, and do not stain for vWF. These results strongly suggest that the tubular structures present in the a-granules of normal porcine platelets correspond to the vWF molecule itself.

Platelet survival studies were performed by the radiochromium method in 280 patients. Although various types of patients were studied, markedly accelerated platelet destruction was encountered only in those with ITP. No thrombocytopenic patient was found with accelerated platelet production and changes in platelet count with therapy reflected corresponding changes in survival. The use of surface scanning showed that patients with significant splenic localization were more suitable candidates for splenectomy that those without this localization.