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A severe coagulopathy is a life-threatening complication of acute promyelocytic leukemia (APL) and is ascribable mainly to the excessive levels of tissue factor (TF) in APL cells regulated in response to the promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha) fusion protein. The underlying molecular mechanisms for this regulation remain ill-defined. With U937-PR9 cell lines stably expressing luciferase reporter gene under the control of different mutants of the TF promoter, both luciferase and ChIP data allowed the localization of the PML/RARalpha-responsive sequence in a previously undefined region of the TF promoter at position -230 to -242 devoid of known mammalian transcription factor binding sites. Within this sequence a GAGC motif (-235 to -238) was shown to be crucial because deletion or mutation of these nucleotides impaired both PML/RARalpha interaction and promoter transactivation. However, EMSA results showed that PML/RARalpha did not bind to DNA probes encompassing the -230 to -242 sequences, precluding a direct DNA association. Mutational experiments further suggest that the activator protein 1 (AP-1) sites of the TF promoter are dispensable for PML/RARalpha regulation. This study shows that PML/RARalpha transactivates the TF promoter through an indirect interaction with an element composed of a GAGC motif and the flanking nucleotides, independent of AP-1 binding.

Platelet factor four (PF4), an inhibitor of endothelial cell proliferation in vitro, inhibits angiogenesis and tumor growth in vivo in experimental animals. The present study was designed to determine whether gene therapy-mediated expression of a form of PF4 lacking 16 amino acids of N-terminus from tumor cells could inhibit angiogenesis and tumor growth in vivo. Two replication-defective recombinant retroviral vectors were constructed. One encodes human PF4 (rRV-PF4) and the other encodes the N-truncated peptide (rRVp17-70). These vectors were then used to transduce KB cells, a human head and neck squamous carcinoma cell line. Expression of PF4 and p17-70 transgenes was confirmed by Western blot analysis. In vitro, both rRV-PF4 and rRVp17-70 were able to inhibit selectively the proliferation of human umbilical vascular endothelial cells (HUVEC) but not KB cells. In vivo activity was assessed by injecting 10(7) KB cells subcutaneously into nude mice and by monitoring subsequent tumor growth, xenograft vascular histochemistry, and animal survival. Viral vector-mediated cDNA transfer of PF4 and p17-70 resulted in inhibiting solid tumors through an anti-angiogenic action in vivo. Our data indicate that targeting tumor angiogenesis using viral-mediated gene transfer of full-length and N-terminal truncated PF4 represents a promising strategy for cancer gene therapy.

Thrombospondin-1 (TSP1) is abundantly secreted during platelet activation and plays a role in irreversible platelet aggregation. A peptide derived from the C-terminal domain of TSP1, RFYVVMWK (RFY) can activate human platelets at least in part via its binding to integrin-associated protein. Although integrin-associated protein is known to physically interact with alphaIIb/beta3, we found that this major platelet integrin had only a partial implication in RFY-mediated platelet aggregation. Accordingly, RFY induced a significant Glanzmann type I thrombasthenic platelet aggregation. The alphaIIb/beta3-dependent part of platelet aggregation induced by RFY was mainly due to secreted ADP and thromboxane A2. In the absence of alphaIIb/beta3 and fibrinogen, RFY stimulated a rapid tyrosine phosphorylation of a set of proteins, including Syk, linker for activation of T cells (LAT) and phospholipase Cgamma2. This signaling pathway was critical for RFY-mediated platelet activation as revealed by the use of pharmacological inhibitors as well as LAT-deficient mouse platelets. Phosphoinositide 3-kinase activation was also required for RFY-mediated platelet aggregation. Our results unravel a new alphaIIb/beta3 and fibrinogen-independent mechanism for platelet aggregation in response to the active peptide from the C-terminal domain of TSP1.

A Glanzmann thrombasthenia variant with a beta3 Ser752-->Pro cytoplasmic domain substitution has platelets that fail to aggregate or bind soluble fibrinogen (Fg) after activation. Despite this, Fg is normally present in the alpha-granules. We have used immunoelectron microscopy to examine the reactivity of Fg with the different pools of alphaIIbbeta3 in the patient's platelets. Immunogold labelling was performed on cryosections using an anti-ligand-induced binding site (LIBS) monoclonal antibody (mAb), which binds to alphaIIbbeta3 only when Fg is bound, or a mixture of two anti-receptor-induced binding site (RIBS) mAbs that specifically recognize receptor-bound Fg. Labelling of the alpha-granule membrane and channels of the surface-connected canalicular system in unstimulated platelets confirmed that the mutated alphaIIbbeta3 retains the capacity to transport Fg. When the patient's platelets were stimulated with ADP in the presence of Fg, as expected there was a much-decreased activation of surface-exposed alphaIIbbeta3. However, thrombin-induced activation was associated with both secretion and a rapid increase in the labelling of internal membrane systems by anti-RIBS and anti-LIBS mAbs, with mobilization of the internal Fg pool. Yet labelling on the surface of the patient's platelets was transient. Our studies implied that alphaIIbbeta3 in platelets may bind fibrinogen in different activation states and that this patient specifically lacked high-affinity binding.

Acetylcholinesterase (AChE) plays a key role in terminating neurotransmission at cholinergic synapses. AChE is also found in tissues devoid of cholinergic responses, indicating potential functions beyond neurotransmission. It has been suggested that AChE may participate in development, differentiation, and pathogenic processes such as Alzheimer's disease and tumorigenesis. We examined AChE expression in a number of cell lines upon induction of apoptosis by various stimuli. AChE is induced in all apoptotic cells examined as determined by cytochemical staining, immunological analysis, affinity chromatography purification, and molecular cloning. The AChE protein was found in the cytoplasm at the initiation of apoptosis and then in the nucleus or apoptotic bodies upon commitment to cell death. Sequence analysis revealed that AChE expressed in apoptotic cells is identical to the synapse type AChE. Pharmacological inhibitors of AChE prevented apoptosis. Furthermore, blocking the expression of AChE with antisense inhibited apoptosis. Therefore, our studies demonstrate that AChE is potentially a marker and a regulator of apoptosis.

This paper describes the molecular defect of the second case of Bernard-Soulier syndrome, initially reported in 1957. Analysis of the patient's platelets by flow cytometry and Western blotting failed to detect surface expression of any of the four subunits of the glycoprotein (GP)Ib-V-IX complex and revealed small amounts of intracellular GPIbalpha, GPIbbeta and GPV but no GPIX. DNA sequencing revealed a novel missense mutation in the GPIX gene which replaced Leu (CTG) by Pro (CCG) at position 7 of the signal peptide. This mutation is, to date, the only known example of a leader sequence defect in Bernard-Soulier syndrome. The change occurred in a prototypic alpha-helical hydrophobic core region, typically enriched in leucine and devoid of proline residues. Co-transfection of GPIXPro7 with normal GPIbalpha and GPIbbeta into Chinese hamster ovary cells reproduced the platelet phenotype, resulting in no detectable GPIX, low intracellular levels of GPIbalpha and GPIbbeta, and an absence of surface expression. This mutation presumably leads to an abnormal conformation and, hence, incorrect insertion of GPIX into the endoplasmic reticulum and/or to defective signal peptide cleavage, both of which are required for correct transport to the cell membrane. This provides further evidence for a critical role of GPIX in controlling biosynthesis of the GPIb-IX complex.

We studied the effect of SR33805, a calcium channel blocker, in vitro on the proliferation of vascular smooth muscle cells (SMC) stimulated by foetal calf serum, basic fibroblast growth factor and platelet derived growth factor, and in vivo with regard to SMC migration and proliferation which occurred following injury of the porcine carotid artery. The intimal lesion was induced by a silasten collar surgically positioned around the carotid artery and by a stenosis reducing blood flow by 50% for 30 days. Animals received SR33805 (5 mg/kg/day) 8 days before the induction of the lesion and up to 30 days after. In vitro, SR33805 inhibited in a dose-dependent manner growth factor-induced proliferation of SMC (0.20

Many chemokines have direct suppressive activity in vitro and in vivo on primitive hematopoietic cells. However, few chemokine-derived peptides have shown a significant activity in inhibiting hematopoiesis. Interestingly, a peptide derived from the 34-58 sequence of the CXC chemokine platelet factor 4 (PF4) produced a 30-40% inhibition of proliferation of murine hematopoietic progenitors (CFU-MK, CFU-GM, and BFU-E) in vitro, at concentrations of 30-60-fold lower than PF4. The aim of the present work was to define the structural parameters and motifs involved in conferring biological activity to the peptide PF4(34-58). Both structural predictions and determinations revealed a new helical motif that was further localized between residues 38 and 46. This helix was necessary for binding of the peptide and for permitting the functional DLQ motif at position 54-56 to activate the putative receptor site. Peptides lacking either the helical or the DLQ motif were devoid of inhibitory activity on the hematopoietic progenitors in vitro. However, among inactive peptides, only those having the helical motif counteracted the inhibition induced by the active peptide PF4(34-58). This suggested that the helix might be required for peptide interactions with a putative receptor site, whereas the DLQ motif would be implicated in the activation of this receptor. These results identify for the first time the dual requirements for the design of chemokine-derived peptides with high suppressive activity on hematopoiesis, as well as for the design of molecules with antagonistic action.

The use of platelet transfusion to ensure the recovery of thrombopoiesis in patients constitutes high-cost support. The identification and cloning of recombinant human thrombopoietin (TPO) and the development of efficient methods of purification of hematopoietic stem cells and progenitor cells have ameliorated the development of strategies of ex vivo expansion of megakaryocyte (MK) progenitor cells and mature MKs. Synergistic combinations of cytokines including TPO, interleukin (IL)-1, IL-3, IL-11, stem cell factor, and FLT-3 ligand induce the ex vivo expansion of colony-forming unit-MK progenitors and MKs from cytokine-mobilized peripheral blood cells, bone marrow, and cord blood CD34+ cells. Depending on the various culture conditions, i.e., combinations of growth factors, initial concentration of CD34+, serum or serum-free cultures, and/or oxygen tensions, the expansion-fold of MKs and their progenitor cells vary greatly. The clinical applications of the reinfusion of ex vivo-generated MK cells have been investigated successfully in cancer patients following high-dose chemotherapy. This review reports the latest information concerning ex vivo expansion of MKs and the current status of clinical trials.

After 35 years of research, a physiological regulator of platelet production has been identified and the recombinant protein is available. With the discovery of thrombopoietin (TPO), its potential use in a wide variety of clinical megakaryocytic and platelet disorders has been expected and clinical trials have been undertaken. To date, the reported encouraging pre-clinical studies indicate that, as with erythropoietin or G-CSF, minimal toxicity can be expected. A potential limiting side-effect of TPO could be the induction of thrombosis. Nevertheless, it is too early to know whether this cytokine will be of major therapeutic importance for patients with life-threatening thrombocytopenia, such as patients undergoing bone marrow transplantation or subjected to a high dose of chemotherapy. Several experimental and clinical studies are still needed to determine the efficacy of TPO in the prevention or the amelioration of bleeding, which is the ultimate goal for the appropriate use of cytokines with haemostatic benefit. Basic and clinical studies on regulators of megakaryocytopoiesis have rapidly progressed. Now, there is no doubt that some of these regulators are effective in correcting haematopoietic disorders of various aetiologies. Studies on negative regulators not only are important to understand the regulation of megakaryocytopoiesis in normal and pathological states but also have a potential clinical application. Some of these regulators have been shown to be effective in the treatment of essential thrombocythaemia and other myeloproliferative disorders. Platelet factor 4 (PF4) and some other chemokines are also capable of protecting progenitor cells from the cytotoxicity of chemotherapeutic drugs. However, detailed investigations are still required to determine the precise mechanism(s) of action of these regulators and to establish the optimal clinical protocols of negative regulators alone or in association with positive regulators for the treatment of various haematological diseases and cancer.

In this study, we examined in detail the interaction of platelet factor-4 (PF-4) with fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) and the effect of PF-4-derived synthetic peptides. We show that a peptide between amino acids 47 and 70 that contains the heparin-binding lysine-rich site inhibits FGF-2 or VEGF function. This is based on the following observations: PF-4 peptide 47-70 inhibited FGF-2 or VEGF binding to endothelial cells; it inhibited FGF-2 or VEGF binding to FGFRs or VEGFRs in heparan sulfate-deficient CHO cells transfected with FGFR1 (CHOFGFR1) or VEGFR2 (CHOmVEGFR2) cDNA; it blocked proliferation or tube formation in three-dimensional angiogenesis assays; and, finally, it competed with the direct association of (125)I-PF-4 with FGF-2 or VEGF, respectively, and inhibited heparin-induced FGF-2 dimerization. A shorter C-terminal peptide (peptide 58-70), which still contained the heparin-binding lysin-rich site, had no effect. Peptide 17-58, which is located in the central part of the molecule, although it does not inhibit FGF-2 or VEGF binding or biologic activity in endothelial cells, inhibited heparin-dependent binding of (125)I-FGF-2 or (125)I-VEGF to CHOmFGFR1 or CHOmVEGFR2 cells, respectively. Shorter peptides (peptides 34-58 and 47-58) did not show any of these effects.

Arsenic trioxide (As2O3) has been demonstrated to be effective for the treatment of acute promyelocytic leukemia (APL) and to inhibit proliferation and produce apoptosis in the APL cell line NB4. To determine if As2O3 might be useful for the treatment of other lineages, we investigated the effects of As2O3 on viability, proliferation, and induction of apoptosis in the megakaryocytic leukemia cell lines HEL, Meg-01, UT7, and M07e. Our results showed that As2O3, at concentrations of 0.1-2.0 microM, causes a dose- and time-dependent inhibition of survival and growth in all four megakaryocytic leukemia cell lines studied. In contrast, As2O3 at similar concentrations had no effects on either viability or growth of the nonmegakaryocytic leukemia cell line HL60 and two human breast cancer cell lines, ZR75 and MCF7. In situ end-labeling of DNA fragments (TUNEL assay) indicated that As2O3, at concentrations of 0.5-2 microM, could significantly induce apoptosis in the aforementioned four megakaryocytic leukemia cell lines, but not in the nonmegakaryocytic HL60, ZR75, and MCF7 cell lines. These results were confirmed using conventional morphologic assessment and the DNA ladder assay. Induction of apoptosis in arsenic-treated Meg-01 and UT7 cells was accompanied by a dose-response decrease of Bcl-2 protein, whereas As2O3 had no effect on this measurement in HL60, ZR75, and MCF7 cell lines. Pertinently, these concentrations of As2O3 produced identical changes in the characteristics of the APL cell line NB4. Collectively, these data demonstrate that As2O3 can selectively inhibit growth and induce apoptosis in megakaryocytic leukemia cell lines. The use of As2O3 for the treatment of malignant megakaryocytic disorders should be considered.

The mechanism of the transient beneficial effect of 1-deamino(8-D-arginine) vasopressin (dDAVP) infusion in the hemostasis of some BSS patients is not fully understood. We have studied the effect of dDAVP infusion in a BSS patient using an ex vivo perfusion system. Additional coagulation and flow cytometry studies were also performed. Prolonged bleeding time (> 30 min) was not affected by dDAVP infusion. However, perfusion experiments performed with low molecular weight heparin anticoagulated blood (which permits the study of fibrin deposition on perfused subendothelium) showed a significant increase in platelet deposition (6.2% before dDAVP infusion; 20.3% after) and fibrin formation. dDAVP infusion also caused an increase in prothrombin consumption compared with base line values (33 vs 46%). Flow cytometry studies of the patients platelets showed no changes in binding of monoclonal antibodies against CD41, CD36, CD62P or CD63. The increase in thrombus formation observed in perfusions may be dependent on FVIII since it could be reproduced by adding purified free or von Willebrand factor (vWf)-associated FVIII to the patient's blood in vitro. The shortening effect of dDAVP on bleeding time observed in some Bernard-Soulier syndrome patients might be related to an increase in factor FVIII levels induced by dDAVP infusion.

The tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been described as an inhibitor of CFU-S entry into DNA synthesis; as a result, its administration can protect mice against lethal doses of cytosine arabinoside (Ara-C). In the present study, we tested the protective effect of AcSDKP on CFU-MK and CFU-GM progenitor cells in mice treated at lower doses of Ara-C more relevant to human clinical situations. Firstly, we report for the first time that in vitro pre-incubation of murine BM MNC with AcSDKP at concentrations of 10(-10) and 10(-9) M for 48 h decreased CFU-MK, in parallel to CFU-GM, progenitor growth. This resulted in an increase of recovery of these progenitors after exposure to Ara-C. Secondly, we tested the effect of AcSDKP on progenitor cells in vivo in different conditions in Ara-C treated mice. We show that the administration of AcSDKP before starting Ara-C treatment resulted in a significant increase in progenitor CFU-GM, CFU-MK and mature MK numbers, 6 and 8 days after the first Ara-C injection. Interestingly, no difference was observed whether AcSDKP was started 24 or 48 h before Ara-C. In a protocol in which AcSDKP was administered for 8 days starting 48 h before Ara-C treatment, the dose did not appear to be critical at least within the range tested (4 vs. 40 micrograms/injection). In addition, the administration of AcSDKP at 64 micrograms/kg per injection for 5 days and stopping it 3 days before the end of Ara-C treatment, i.e. five instead of eight applications, further increased its protective effect. Thus our results demonstrate protective effect of AcSDKP for progenitors during a fractionated protocol of Ara-C treatment and indicates an importance of the dose and the schedule of administration of AcSDKP in designing future clinical trials.

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 microg/mL (half maximum inhibition at 0.6 micro/mL), and proliferation was completely inhibited at 2 microg/mL. At this concentration, PF-4 reduced internalization of 125I-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.

Platelet factor 4 (PF4) has been recognized as an inhibitor of myeloid progenitors. However, the mechanism of action of this chemokine remains poorly understood. The present study was designed to determine its structure/function relationship. A series of peptides overlapping the C-terminal and central regions of PF4 were analyzed in vitro for their action on murine hematopoietic progenitor growth to assess the minimal sequence length required for activity. The peptides p17-58 and p34-58 possessed an increased hematopoietic inhibitory activity when compared with PF4, whereas the shorter peptides p47-58 and p47-70 were equivalent to the native molecule and the peptide p58-70 was inactive. The PF4 functional motif DLQ located in 54-56 was required for the activity of these peptides. The peptide p34-58 impaired to a similar extent the growth of colony-forming unit-megakaryocyte (CFU-MK) as well as burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte-macrophage (CFU-GM), whereas PF4 was more active on CFU-MK. In the experiments using purified murine CD34(+) marrow cells, statistically significant inhibition induced by p34-58 was shown at concentrations of 2.2 nmol/L or greater for progenitors of the three lineages, whereas that induced by PF4 was seen at 130 nmol/L for CFU-MK and 650 nmol/L for CFU-GM and BFU-E, indicating that the p34-58 acts directly on hematopoietic progenitors and its activity is approximately 60- to 300-fold higher than PF4. The p34-58, unlike PF4, lacked affinity for heparin and its inhibitory activity could not be abrogated by the addition of heparin. In addition, an antibody recognizing p34-58 neutralized the activity of p34-58 but not whole PF4 molecule. These results demonstrate that PF4 contains a functional domain in its central region, which is independent of the heparin binding properties, and provide evidence for a model of heparin-dependent and independent pathways of PF4 in inhibiting hematopoiesis.

Chemokines are a large family of cytokines that act not only as immune and inflammatory regulators but also as regulators of hematopoiesis. Two major subfamilies of chemokines are distinguished on the basis of whether the first two cysteines are separated by a single residue (CXC) or three residues (CX3C) or they are adjacent (CC) or there is a single C. The Macrophage Inflammatory Protein 1 alpha (MIP-1 alpha), which belongs to CC family is a powerful inhibitor of hematopoisis in vitro and in vivo. The sub-family CXC comprises two main groups. The first sub-group includes the ELR chemokines, in which interleukin-8 (IL-8) is the most prototypic and possesses suppressive activities on hematopoiesis. Platelet Factor 4 (PF4) belongs to the sub-group of non-ELR CXC chemokines. PF4 acts as an inhibitor of hematopoiesis, particularly of the megakaryocytopoiesis. Recently, it has been shown that a peptide of PF4, 34-58 which does not contain the site of heparin binding, is able to inhibit the growth of hematopoietic progenitors in vitro, providing evidence for a model of heparin dependent and independent pathways of PF4 action on hematopoiesis. PF4 can reduce the chimiosensitivity of hematopoietic cells in mice treated by the cytotoxic drug 5-Fluorouracyl, suggesting a potential clinical application of PF4 in cancer therapy.

A computer-assisted automatic image procedure was karyocytopoiesis in culture. This analysis system was based on acetylcholinesterase staining, a specific staining for murine bone marrow megakaryocytes, and an image capturing instrument with a computer program. Two kinds of routine megakaryocyte culture methods were used, the plasma clot and the serum-free agar systems. A comparison between manual counting and the instrument was made. The image analysis software was able to distinguish between megakaryocytes (MK) at different stages of maturation. The results show that this analysis system can simultaneously detect not only the number of megakaryocytes and their colonies in each dish, but also the surface area of individual megakaryocytes. In addition, this analysis system functions automatically 24 hours a day and the results obtained are reproducible. Using this system, we have confirmed previous observations that thrombopoietin (TPO) and heparin stimulate both proliferation and maturation of megakaryocytes. In addition, we found that platelet factor 4 (PF-4) significantly reduced the number of megakaryocytes but not their cell surface area, whereas TGFbeta1 decreased both number and surface area of megakaryocytes, suggesting that PF4 and TGFbeta1 negatively regulate megakaryocytopoiesis by different mechanisms. We noticed that megakaryocytes grown under agar culture conditions regularly had an increased size in comparison with those grown in a plasma clot system, which may be an indication that the plasma clot culture media contains an inhibitor(s) of megakaryocyte maturation. Our data indicate that this image analysis system, in addition to its automatic and reproducible features, is more efficient and allows detection of more parameters than routine manual microscopic detection.

Mouse embryonic stem (ES) cells transfected with a 1.7 kb cDNA of porcine transforming growth factor type beta1 (TGFbeta1), known as ES-T cells, were found to be able to differentiate in vitro into cystic embryonic bodies (EBs) with outspread tubular structures. Morphological analysis using light, phase-contrast and electron microscopes revealed that in culture, the EBs of ES-T cells initially developed some flat endothelial-like cells which further proliferated and migrated to form thread structures. At 8-10 days after EB formation, these thread structures further developed into net-like and tubular structures connecting directly to EBs. Immunofluorescent assays using antibodies against Flk-1 and von Willebrand factor (vWF) indicated that these net-like and tubular structures of ES-T cells consisted of vascular endothelial cells. Further analysis by RT-PCR revealed that the EBs with tubular structures expressed the mRNA of other markers of vascular endothelial cells, including VE-cadherin and platelet-endothelial cell adhesion molecule (PECAM). Cells of hematopoietic origin were not detected on the outside of EBs by immunostaining using several antibodies specific for granulocytes, macrophages and lymphocytes as well as by benzidine staining for erythroid cells on the outside of EBs. Our data demonstrates that the transfer of TGFbeta1 into ES cells results in a significant vasculogenesis without concomitant hematopoiesis. ES-T cells could therefore provide an excellent model for studying blood vessel formation and vasculogenic and hematopoietic interactions.

We have recently reported that platelet factor 4 (PF4), a megakaryocyte-platelet protein, is a potent inhibitor of human and murine megakaryocytopoiesis. In addition, PF4 accelerated the recovery of the marrow precursor cells in 5-fluorouracil (5-FU) treated mice. We show in this study that a slight modification of the C-terminal peptide related to PF4 (C13-24DE), which was previously reported as the carboxy terminal region of PF4 implicated in PF4 inhibitory activity, is also able to significantly increase murine high proliferating-potential-colony forming cells (HPP-CFC), colony-forming-unit megakaryocyte (CFU-MK) and colony-forming unit granulocyte-macrophage (CFU-GM) progenitor number, eight days after 5-FU administration, when it was given intraperitoneally twice a day (200 micrograms/kg/inj) prior to 5-FU administration (150 mg/kg). Furthermore, the C13-24DE pretreatment enhanced both the number and the diameter of single megakaryocyte (MK) by day 8. These data indicate that the C13-24DE peptide related to PF4 accelerated the in vivo recovery of stem cells, progenitors (CFU-GM, CFU-MK) and single MK after 5-FU treatment and may have a hemoprotective effect against chemotherapeutic agents.

Activation of the mitogen-activated protein (MAP) kinase pathway in nucleated cells is dependent on both growth factor receptors and integrins engaged in cell adhesion. Human platelets are an interesting model for studying cell adhesion and the involvement of integrin engagement on extracellular signal-regulated kinase (ERK) activation, independently from the nuclear-DNA signal pathway. Maximal phosphorylation and activity of ERK2 occurred late during thrombin-induced platelet aggregation (90 s and later), an alphaIIbbeta3 integrin-dependent event. Surprisingly, alphaIIbbeta3 inhibition by the RGDS ligand peptide, or (Fab')2 fragments of the AP-2 monoclonal antibody, resulted in a 2-fold enhancement in ERK2 phosphorylation and activity. A similar 2-fold enhancement of ERK2 activation was observed in thrombasthenic platelets which are defective in alphaIIbbeta3 and do not aggregate. This suggests that ERK2 activation in thrombin-induced platelet aggregation is dependent on thrombin rather than on alphaIIbbeta3 and is down-regulated by alphaIIbbeta3 engaged in ligand (fibrinogen) binding and/or aggregation. Finally, in the absence of stirring which allows fibrinogen binding to alphaIIbbeta3 but prevents aggregation, ERK2 was again overactivated. This overactivation appears to be consecutive to inhibition of aggregation itself and to alphaIIbbeta3 ligand binding. We conclude that in platelets, alphaIIbbeta3 engaged in aggregation down-regulates thrombin-induced ERK2 activation. To our knowledge, this is the first report of a down-regulation of the MAP kinase pathway by integrin engagement.

Tyrosine phosphorylation of a number of platelet proteins is dependent on platelet integrin alphaIIb beta3 (also termed GPIIb-IIIa) and its engagement in aggregation. For instance, in type I thrombasthenic platelets, which lack alphaIIb beta3 and do not aggregate, several substrates are either poorly or not phosphorylated. We have compared thrombasthenic platelets of type I, type II (15% alphaIIb beta3, functional), and variant type (50% alphaIIb beta3, no fibrinogen binding). The platelets from the three patients exhibited the same low tyrosine phosphorylation profiles, confirming the key role of functional alphaIIb beta3 in initiating protein tyrosine phosphorylation. We noted that in addition to the characteristic absence of the 100 to 105 kD doublet, a 77 to 80 kD doublet and to a lesser extent a 64-kD band, exhibited low phosphorylation kinetics, but with normal initial phosphorylation rates (up to 60 seconds). Similar results were obtained by inhibition of thrombin aggregation of control platelets by alphaIIb beta3 antagonists (the RGDS peptide or the monoclonal antibody 10E5), or in the absence of stirring (fibrinogen binding, but no aggregation). These results suggest that tyrosine phosphorylation of the 77 to 80 kD doublet, identified by immunoprecipitation as the cytoskeletal protein cortactin, and the 64 kD band are dependent both on thrombin activation during early steps and on the late steps of alphaIIb beta3 engagement in aggregation. Implications as to involvement of step-specific kinase and/or phosphatase activities are discussed.

The relative involvement of the glycoprotein (GP) Ib/IX-von Willebrand factor (vWF) axis and GPIIb/IIIa in thrombus growth at high shear rates was assessed and compared by testing the pharmacological effects of VCL, a recombinant GPIb-binding fragment of vWF (residues 504-728), aurintricarboxylic acid (ATA), which binds to the 509-695 disulfide loop of vWF, and lamifiban, a specific synthetic GPIIb/IIIa antagonist. In vivo, their effects were evaluated in guinea pig mesenteric arteries, in a model of a laser-induced cyclic thrombotic process, and ex vivo, at a shear rate of 1800 s(-1), in a capillary perfusion chamber model, in which collagen-adherent platelets are exposed to nonanticoagulated guinea pig blood. In vivo, VCL, ATA, and lamifiban administered 2 minutes after intimal injuries stopped thrombus growth, prevented the cyclic thrombotic process, and induced gradual thrombus dissolution. Ex vivo, at 1800 s(-1), collagen exposure to untreated blood for 2 minutes, 4 minutes, or two consecutive periods of 2 minutes each resulted in similar platelet adhesion, 56%, 59%, and 61%, respectively, with an average thrombus volume of 6, 19, and 17.5 microm3/microm2, respectively, without any fibrin formation. This indicated that the two consecutive perfusions did not affect the dynamic process of thrombus growth. When collagen-adherent platelets deposited after the first 2-minute perfusion were perfused for 2 minutes with VCL-, ATA-, or lamifiban-treated blood, thrombus growth was prevented and platelet adhesion remained unchanged, but fibrin formation increased on and around the predeposited platelets. These results suggest that both the GPIb/IX-vWF axis and GPIIb/IIIa are involved in in vivo platelet-to-platelet interactions at high shear rates in the guinea pig.

The effects of platelet factor 4 (PF4) on the viability and chemosensitivity of normal hematopoietic cells and cancer cell lines were studied to determine the mechanisms whereby PF4 functions as either an inhibitor or a protector and to evaluate its clinical significance. Two other chemokines, interleukin-8 (IL-8) and neutrophil-activating peptide-2 (NAP-2), were also studied in comparison to PF4. Using a tetrazolium salt assay for cell viability, we observed that PF4 at 1 to 50 microg/mL supported the viability of normal human bone marrow cells. Approximately 45% of cells cultured for 48 hours survived, whereas 80% or more survived in the presence of PF4 5 microg/mL. PF4 also supported the viability of CD34+ cord blood (CB) cells and protected them from apoptosis induced by transforming growth factor beta1 (TGFbeta1) and cytotoxic drugs. Pretreatment of CD34+ cells by PF4, but not by TGFbeta1, caused an increase in the number of megakaryocyte colonies after these cells were replated in secondary cultures. Flow cytometry analysis showed that when CD34+ cells were preincubated with PF4 or TGFbeta1 for 12 days in hematopoietic growth factor-rich medium, an increased number of remaining CD34+ cells was observed only for PF4-treated cells. Furthermore, PF4 significantly reduced the chemosensitivity of bone marrow cells, as shown by its ability to increase the 50% inhibition concentration (IC50) of several cytotoxic agents. Like PF4, IL-8 and NAP-2 at 0.1, 0.6, and 1 microg/mL supported the survival of myeloid progenitors, including colony-forming units granulocyte, erythroblast, monocyte, megakaryocyte (CFU-GEMM), CFU-megakaryocyte (CFU-MK), CFU-granulocyte/macrophage (CFU-GM), and burst-forming units-erythroblast (BFU-E), and reduced their sensitivity to the toxicity of etoposide (ETP). Protamine sulfate at 1 to 100 microg/mL showed no such activity of PF4. Interestingly, the three chemokines failed to affect significantly the viability and chemosensitivity of three leukemic and two other tumor cell lines. Based on these results, we conclude for the first time that PF4 and IL-8 and NAP-2 support the survival of normal hematopoietic precursors and protect them from the toxicity of chemotherapeutic agents. Because such activities are unique to normal hematopoietic cells but not to the cancer cell lines evaluated, a potential clinical application of these molecules in the treatment of cancer is suggested.

The Bernard-Soulier syndrome (BSS) is characterized by thrombocytopenia with giant platelets, a prolonged bleeding time with defective platelet adhesion to the subendothelium related to a defect in platelet membrane glycoprotein Ib (GPIb) and a decreased prothrombin consumption. The mechanism of the latter abnormality remains unknown. In this study, we showed that this defect was corrected by the addition of purified human factor VIII (FVIII) to blood from four patients with BSS. The correction of prothrombin consumption was almost complete at concentrations between 1.5 and 3 IU/ml of FVIII procoagulant activity (VIII:C) and partially abolished by a monoclonal antibody which neutralizes VIII:C. This correction was specific for FVIII and was not observed after addition of purified human FIX. It was obtained, in the same magnitude range, with FVIII complexed to von Willebrand factor (vWF) but not with free vWF. These data provide a new insight into the knowledge of the physiological interaction between the platelet membrane and the vWF-FVIII complex facilitating plasma coagulation activation and may lead to helpful therapeutic advances.

SHP-1 is a cytoplasmic protein tyrosine phosphatase predominantly expressed in hematopoietic cells. Upon thrombin stimulation of human platelets, SHP-1 is rapidly phosphorylated on both serine and tyrosine residues, and becomes associated with the cytoskeleton, where it could participate in the formation of multiprotein signalling complexes. In order to discriminate between signalling events occurring downstream of G-protein-coupled thrombin receptor and those subsequent to integrin alpha IIb beta 3 engagement, SHP-1 behaviour was examined in platelets from two patients lacking integrin alpha IIb beta 3 (Glanzmann's thrombasthenia). Upon thrombin stimulation, phosphorylation of SHP-1 occurred normally in thrombasthenic platelets, whereas association with the cytoskeleton was abolished. Moreover, inhibition of normal platelet aggregation with the tetrapeptide arg-gly-asp-ser (RGDS) which impairs fibrinogen binding to integrin alpha IIb beta 3, did not alter significantly SHP-1 phosphorylation. It is concluded that SHP-1 phosphorylation is not a consequence of integrin signalling but might rather occur downstream of thrombin receptor and heterotrimeric G-proteins.

In vivo effects of platelet factor 4 (PF4) and tetrapeptide N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) on haemopoietic progenitors were studied in mice treated with 5-fluorouracil (5-FU). The mice were injected with PF4 (40 micrograms/kg) or AcSDKP (4 micrograms/kg) twice at 6 h intervals, and 20 h after the second injection they were given one injection of 5-FU (150 mg/kg). 6, 8 and 13 d later the high proliferative potential-colony forming cell (HPP-CFC), burst-forming unit erythroid (BFU-E), colony forming unit granulocyte-macrophage (CFU-GM) colony forming unit megakaryocyte (CFU-MK), and megakaryocytes (MK) were examined. The results showed that the administration of PF4 or AcSKDP resulted in a significant increase in the number of HPP-CFC on days 6-8 and BFU-E and CFU-GM on day 8 when compared to 5-FU alone. Furthermore, PF4 was found to increase significantly the number of CFU-MK and MK on day 8, which was not observed with AcSDKP. However, both molecules had no obvious effect on peripheral blood cells. These data indicate that PF4 or AcSDKP accelerate the recovery in vivo of HPP-CFC, CFU-GM and BFU-E after 5-FU treatment but their effect may be different on megakaryocytic progenitors and suggests that both molecules may have a haemoprotective effect against chemotherapeutic agents.

Antithrombotic activity of two recombinant GPIb-binding fragments of vWF, RG12986 (residues 445-733), and VCL (residues 504-728), were assessed in an ex vivo capillary perfusion chamber exposing human type III collagen to native nonanticoagulated guinea pig blood. Platelet adhesion and thrombus formation were evaluated by computer assisted morphometry for two shear rates (650 and 1800 s-1) and for two perfusion times (1.5 and 4 min). At 1800 s-1 and 4 min of perfusion, platelet adhesion decreased from 63 +/- 7% for control, to 46 +/- 4% for 20 mg/kg RG12986, and to 29 +/- 5% for 4 mg/kg VCL, and the mean thrombus height dropped from 40 +/- 8 microns to 24 +/- 3 microns and 7.5 +/- 1 microns, respectively. The two doses did not change bleeding time values. Our results suggest that guinea pig blood and the circular perfusion chamber represent a good model for the evaluation of limited amount of GPIb/IX-vWF axis inhibitors.

We have previously reported that heparin is capable of stimulating in vitro and in vivo megakaryocytopoiesis in mice and has a thrombopoietic effect when given in chronic immune thrombocytopenic purpura and that heparin and several other glycosaminoglycans (GAGs) promote the growth of human megakaryoblastic cell lines in the presence of serum. We show here that GAGs, including heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronic acid (HA), also stimulate in vitro growth of murine megakaryocyte progenitors and augment the diameter of individual megakaryocytes in the presence of serum. However, in a serum-free agar system, the GAGs alone had no effect on megakaryocyte colony formation, suggesting that GAGs cooperate with some serum factor(s) to exert their activity. We also show that heparin significantly potentiates the megakaryocytopoietic activity of C-Mpl ligand and interleukin (IL)-6 but not IL3, GM-CSF, SCF, and Epo. In addition, the GAGs significantly neutralize the inhibitory action of platelet factor 4 (PF4) and transforming growth factor beta 1 (TGF beta 1) on megakaryocyte colony growth. These results demonstrate a stimulating activity of GAGs on megakaryocytopoiesis by modifying the activity of several growth-regulating factors.

Cross-linking of fibrinogen at its COOH-terminal gamma chain cross-linking site occurs in the presence of factor XIIIa due to self-association at a constitutive D domain site ("gammaXL"). We investigated the contribution of COOH-terminal regions of fibrinogen Aalpha chains to the gammaXL site by comparing the gamma chain cross-linking rate of intact fibrinogen (fraction I-2) with that of plasma fraction I-9, plasmic fraction I-9D, and plasmic fragment D1, which lack COOH-terminal Aalpha chain regions comprising approximately 100, approximately 390, and 413 residues, respectively. The cross-linking rates were I-2 > I-9 > 1-9D = D1, and indicated that the terminal 100 or more Aalpha chain residues enhance gammaXL site association. Fibrinogen Dusart, whose structural abnormality is in the COOH-terminal "alphaC" region of its Aalpha chain (Aalpha R554C-albumin), is associated with thrombophilia ("Dusart Syndrome"), and is characterized functionally by defective fibrin polymerization and clot structure, and reduced plasminogen binding and tPA-induced fibrinolysis. In the presence of XIIIa, the Dusart fibrinogen gamma chain cross-linking rate was about twice that of normal, but was normalized in proteolytic fibrinogen derivatives lacking the Aalpha chain abnormality, as was reduced plasminogen binding. Electron microscopy showed that albumin-bound Dusart fibrinogen "alphaC" regions were located in the vicinity of D domains, rather than at their expected tethered location near the fibrinogen E domain. In addition, there was considerable fibrinogen aggregation that was attributable to increased intermolecular COOH-terminal Aalpha chain associations promoted by untethered Dusart fibrinogen aC domains. We conclude that enhanced Dusart fibrinogen self-assembly is mediated through its abnormal alphaC domains, leads to increased gammaXL self-association and gamma chain cross-linking potential, and contributes to the thrombophilia that characterizes the "Dusart Syndrome."

Mechanisms of the action of platelet factor 4 (PF4) on the growth of megakaryocyte (MK) progenitor cells in CD34+ cord blood (CB) cells were studied in comparison with transforming growth factor beta1 (TGFbeta1). Development of MK from CD34+ CB cells in both plasma clot culture and liquid culture was significantly inhibited by either purified human PF4 and by recombinant human TGFbeta1. Inhibition of MK colony formation by PF4 was reversible because CD34+ cells preincubated with PF4 could regenerate colonies after washing and replating into secondary cultures. In contrast, TGFbeta1-preincubated CD34+ cells gave rise to few colonies following replating. Moreover, incubation of CD34+ cells with PF4 in liquid culture caused the increased number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells. In addition, PF4-preincubated CD34+ cells exhibited a higher potential in MK colony formation in the presence of 5-fluorouracil (5FU). These results demonstrate that both PF4 and TGFbeta1 inhibit MK development from CD34+ CB cells by different mechanisms, and suggest that PF4, unlike TGFbeta1, exerts its inhibitory effect on the growth of the target cells in a reversible manner which results in a preservation of a more immature and 5FU-resistant cell population.

We have previously shown that platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) inhibit the growth of the human erythroleukemia cell line (HEL). We further studied the effect of PF4, beta-TG, and various related peptides on human leukemic lineages to determine the specificity and the relationship between the inhibitory activity and the molecular structure of PF4. The results showed that PF4 and beta-TG had an inhibitory activity on the megakaryocytic growth. Furthermore, peptides corresponding to the 1-24 and 13-24 residues but not to the 16-24 residue of the PF4 C-terminal region, the 21-29 and 20-28 C-terminal region of beta-TG and IL-8, inhibited only the megakaryocytic cell growth. Interestingly, when Gln and Asn located at positions 15 and 24, respectively, of the PF4 C-terminal region were replaced by Glu and Asp (C13-24DE), an increase in the inhibitory activity was observed. Moreover, the 13-24 monomeric form (13-24M) and modified form (13-24A), where a cysteine in C-terminal position 19 was substituted by arginine, were no longer active. These results suggest that the inhibitory activity of PF4 and its related peptides might be localized in their 13-24 C-terminal region and that a dimeric structure seems to be necessary to exert inhibitory activity.

Ultrastructural perturbations resulting from defects in polymerization of fibrinogen Dusart, a congenital dysfibrinogenemia with the amino acid substitution A alpha 554 arginine to cysteine, were investigated by a variety of electron microscope studies. Polymerization of this mutant fibrinogen on addition of thrombin is impaired, producing clots with decreased porosity and increased resistance to fibrinolysis, resulting in thrombotic complications in the family members with this dysfibrinogenemia. Electron microscopy of rotary-shadowed individual molecules revealed that, in contrast to control fibrinogen, most of the alpha C domains of fibrinogen or fibrin Dusart appeared to be free-swimming appendages that do not exhibit intra- or intermolecular interactions either with each other or with the central domains. The location of albumin on the alpha C domains was demonstrated by electron microscopy using anti-albumin antibodies. Electron microscopy of negatively contrasted fibrin Dusart fibers indicated that they were less ordered than control fibers and had additional mass visible. Electron microscopy of freeze-dried, unidirectionally shadowed fibers showed that they were twisted with a shorter pitch. Scanning electron microscopy revealed that intact clots were made up of thin fibers with many branch points and very small pore sizes. The viscoelastic properties of Dusart fibrin clots measured with a torsion pendulum indicated a marked increase in stiffness consistent with the structural observations.

To evaluate the involvement of the glycoprotein (GP) IIb/IIIa-dependent process in platelet deposition and thrombus growth on capillaries coated with human type III collagen, the effects of incremental doses of Lamifiban, a potent specific synthetic GPIIb/IIIa antagonist, were studied in ex vivo capillary perfusion chambers using guinea pig blood. In this model, nonanticoagulated blood was perfused for 4.5 minutes at three shear rates: 100, 650, and 1600 s-1. Platelet deposition was quantified by computer-assisted morphometry and expressed as platelet adhesion (percentage of capillary surface covered with spread and contact platelets and platelets implicated in thrombus), mean thrombus height, and total thrombus cross-sectional area. In control untreated guinea pigs, platelet adhesion and thrombus height were 63% and 2.5 microns at 100 s-1, 60.5% and 13.8 microns at 650 s-1, and 45% and 28.1 microns at 1600 s-1, respectively. At 100 s-1, Lamifiban had no effect on platelet deposition at any of the three doses administered to the guinea pigs (0.3, 1, and 3 mg/kg). At 0.3 mg/kg and shear rates of 650 and 1600 s-1, Lamifiban had no effect on platelet adhesion or thrombus size, but at 1 and 3 mg/kg and shear rates of 650 and 1600 s-1, it significantly reduced thrombus size. At 1600 s-1, 1 mg/kg Lamifiban significantly increased platelet adhesion from 45% to 62.5%, whereas at 3 mg/kg it induced a significant overall decrease from 45% to 25% and qualitatively increased the ratio of contact to spread platelets. These data suggest that at high shear rates, GPIIb/IIIa participates in platelet spreading and that there is a balance between platelet involvement in adhesion to the thrombogenic surface and the growth of the already formed thrombus. This indicates that important clinical implications of an optimal therapeutic degree of GPIIb/IIIa antagonism could be expected.

Negative regulation of megakaryocytopoiesis is a complex process involving various cytokines. One of these cytokines is platelet factor 4 (PF4), a megakaryocyte/platelet specific protein. PF4 and a carboxy-terminal peptide related to PF4 have been reported to inhibit human and murine megakaryocytopoiesis. The growth of several megakaryoblastic cell lines: human erythroleukaemia cell line (HEL). Meg-01 and Dami, was also inhibited by PF4 and a 13-24 carboxy-terminal peptide related to PF4. We report that peptides corresponding to the 1-24 and 13-24 but not 1-13 carboxy-terminal region of PF4 inhibit murine megakaryocytopoiesis both in vivo (5 micrograms/inj) and in vitro (2.5 and 5 micrograms/ml). Moreover, such an inhibitory activity of PF4-related peptides is abrogated by heparin (5 IU/dish). These overall data indicate that carboxy-terminal PF4-related peptides retain the inhibitory effect of PF4 on both murine single MK and CFU-MK in vivo and in vitro by acting on an early stage of megakaryocytopoiesis and strongly suggest that the inhibitory activity of the multi-functional PF4 might be localized in a short carboxy-terminal region which might include, in part, the PF4 heparin binding domain.

Megakaryocytopoiesis concerns the commitment of hematopoietic stem cells towards the megakaryocyte lineage, the proliferation and maturation of megakaryocyte progenitors, leading to platelet formation. Normal megakaryocytopoiesis requires an equilibrium between positive and negative regulators. Thrombopoietin, recently cloned, is the main growth factor for the megakaryocyte lineage, enhancing all the steps of megakaryocyte development: proliferation, maturation, ploidisation, platelet formation. Several other factors have a direct (interleukin 3, 6, 11, 13, GMCSF, erythropoietin) or indirect (interleukin 1, stem cell factor) positive effect. Factors inhibiting megakaryocytopoiesis are synthetized by the megakaryocytes themselves (platelet factor 4, transforming growth factor beta), or have many other effects (alpha-interferon, thrombin) or correspond to a novel molecule (anagrelide). Heparin and other glycosaminoglycans positively modulate megakaryocytopoiesis by acting synergistically with some growth factors and by neutralizing some inhibitors. These progresses in the knowledge of normal and pathological megakaryocytopoiesis should lead to considerable therapeutic advances.

We have previously shown that platelet factor 4 (PF 4) is a potent inhibitor of megakaryocytopoiesis and that it may protect stem cells from 5-fluorouracil (5-FU) cytotoxicity. In the present work, the effects of human PF 4 on megakaryocyte (MK) growth from human CD34+ cord blood (CB) cells were studied in comparison with transforming growth factor beta 1 (TGF-beta 1). Development of MK from CD34+ cells in both plasma clot culture and liquid culture was significantly inhibited by PF 4 (5 micrograms/ml) and TGF beta 1 (1 ng/ml). Inhibition of cell growth by PF 4 was reversible judging from the fact that the CD34+ cells preincubated with PF 4 could regenerate colonies after washing and replating into the cultures. By contrast, TGF-beta 1 pretreated CD34+ cells gave rise to few colonies following replating. Moreover, incubation of CD34+ cells with PF 4 in liquid culture caused an increase in the number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells, and exhibited greater capacity to form MK colonies than control after the treatment of 5-FU. In vivo in mice, twice injections of PF 4 at 40 micrograms/kg with an interval of 6 h followed by one injection of 5-FU at 150 mg/kg resulted in a significant increase in the number of colony-forming cells with high proliferative potential (HPP-CFC) and colony-forming unit-megakaryocyte (CFU-MK) in bone marrow. In exponentially growing human erythroleukemia cells (HEL), the addition of PF 4 prolonged cell cycle progression and therefore resulted in an increased cell population in S phase, as determined by flow cytometric analysis. Different from PF 4, TGF-beta 1 blocked more cells in G 1 phase. These results demonstrate that PF 4 and TFG-beta 1 inhibit MK development from CD34+ CB cells by different mechanisms and suggest that PF 4, unlike TGF-beta 1, exerts its inhibitory effect on cell growth in a reversible and S phasespecific manner by which it protects stem cells and MK progenitor cells from 5-FU cytotoxicity.

Development of megakaryocyte (MK) from CD34+ cord blood (CB) cells in both plasma clot culture and liquid culture was significantly inhibited by human platelet factor 4 (PF4) and human transforming growth factor beta 1 (TGF beta 1). Inhibition of cell growth by PF4 was reversible judging from the fact that the CD34+ cells preincubated with PF4 could regenerate colonies after washing and replating into the cultures. By contrast, TGF beta 1-pretreated CD34+ cells gave rise to few colonies following replating. Moreover, incubation of CD34+ cells with PF4 in liquid culture caused an increase in the number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells, and exhibited greater capacity to form MK colonies than control after the treatment of 5-FU. In vivo in mice, twice injections of PF4 at 40 micrograms/kg resulted in a significant increase in the number of colony-forming cells with high proliferative potential (HPP-CFC) and colony-forming unit-megakaryocyte (CFU-MK) in bone marrow. In exponentially growing human erythroleukemia cells (HEL), the addition of PF4 prolonged cell cycle progression and therefore resulted in an increased cell population in S phase, as determined by flow cytometric analysis. Different from PF4, TGF beta 1 blocked more cells in G1 phase. These results demonstrate that PF4 and TGF beta 1 inhibit MK development from CD34+ CB cells by different mechanisms and suggest that PF4, unlike TGF beta 1, exerts its inhibitory effect on cell growth in a reversible and S phase-specific manner by which it protects stem cells and MK progenitor cells from 5-FU cytotoxicity.

We have previously shown that basic fibroblast growth factor (bFGF) stimulates megakaryocytopoiesis and granulopoiesis in vitro and that normal haematopoietic cells and several leukaemic cell lines express FGF receptors. In this paper, we demonstrate by reverse transcriptase-mediated polymerase chain reaction (RT-PCR) that bFGF mRNA is expressed in two leukaemia cell lines with megakaryocytic features (Meg-01 and K562), in two lymphocytic cell lines (Hut 78 and CA) and in normal human peripheral blood mononuclear cells. In addition, the conditioned media of Meg-01, but not Dami, contained a potent fibroblast-stimulating activity which could be neutralized by bFGF antibodies. Furthermore, bFGF antibody significantly inhibited the autocrine growth of Meg-01 cells in vitro. However, we could not detect cell-associated 18 kDa bFGF or HMW bFGF by immunofluorescence, immunoprecipitation or Western blotting. These data indicate that bFGF is expressed by certain haematopoietic cells and support further a role of this FGF prototype in haematopoiesis.

Effects of recombinant human interleukin (IL)-13 on in vitro haemopoiesis from non-adherent mononuclear cells (NAMC) or highly enriched CD34+ cells of human cord blood (CB) were studied. IL-13 significantly increased megakaryocyte (MK) colony formation from either NAMC or CD34+ cells cultured in a plasma clot system supplemented with aplastic anaemia serum (AAS) and phytohaemagglutinin-stimulated human peripheral blood leucocyte-conditioned medium (PHA-LCM) in a dose-dependent manner. Experiments using a modified plasma clot culture, in which normal AB serum and various cytokines were added to replace AAS and PHA-LCM, demonstrated an increased MK colony number in the presence of IL-13, especially in combination with IL-3. However, IL-13 had no stimulatory effect, but rather a slight inhibitory effect in some cases on granulocyte-macrophage (GM) colony formation in both plasma clot cultures. Furthermore, the growth of GM progenitor cells in a methylcellulose culture system in the presence of IL-3, GM-CSF, Epo, G-CSF or in combination was significantly inhibited by the addition of IL-13. On the other hand, high concentrations (100 ng/ml) of IL-13 were needed to cause a slight inhibition on the growth of BFU-E-derived colonies under the same methylcellulose culture. These results indicate that IL-13, alone and synergistically with the effect of IL-3, promotes MK colony formation, but it inhibits the growth of GM and erythroid progenitor cells in vitro.

Our previous in-vitro and in-vivo studies showed that heparin enhanced murine and human megakaryocytopoiesis. 20 patients with chronic immune thrombocytopenic purpura were randomly divided into two groups and given 10 mg per day of prednisone for 30 days, for haemostatic purposes. One group received in addition heparin (1250 IU twice a day subcutaneously for 30 days). From day 10, a significant increase in platelet count was observed in eight of the ten patients treated with heparin (p < 0.05), with return to the initial value after heparin cessation in six of the responders. These data demonstrate the effectiveness of heparin and suggest its use or that of other related compounds for therapy of chronic immune thrombocytopenic purpura.

This paper is an overview of the progress made in the field of platelet-vessel wall interaction during the past three quarters of a century. (I) "Prehistoric" era (1918-1948): description of Thrombasthenia by 3 European clinicians (Glanzmann, Switzerland, 1918), Hereditary Pseudohemophila (von Willebrand, Finland 1926) and Congenital Hereditary Platelet Dystrophy (Bernard and Soulier, France 1948). (II) Physiopathological era (1957-1972) based on the understanding of these 3 disorders: abnormality of platelet adhesion to the vessel wall in von Willebrand (vW) and Bernard Soulier syndrome (BSS), abnormality of platelet aggregation in thrombasthenias (GT). Breakthrough was made by I.M. Nilsson et al in vWD in Aåland Islands with the recognition of a plasmatic anomaly, later confirmed by Cornu et al to be different from factor VIII deficiency. (III) The third and crucial development (glycoprotein era) (1974-1981) came from the discovery by Nurden & Caen (1974-1975) of GPIIb-IIIa defect in GT and of GPIb-IX in BSS. Polyclonal antibodies against GPIIb-IIIa and GPIb-IX inhibit clearly ex vivo platelet aggregation and adhesion respectively. On everted rabbit subendothelium platelet, adhesion was abnormal in BSS whichever shear stress and at high shear stress in vWD. (IV) The molecular biology era (1984-1995) with the exquisite recognition of gene or protein anomalies in the above mentioned disorders together with the cloning of the 3 genes [for vW factor (vWF) (GPIb-IX, GPIIb, and GPIIIa]. (V) We are at the dawn of the more crucial era, the antithrombotic therapy acting either on the GPIb-vWF complex or on the GPIIb-IIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

KRDS, a tetrapeptide from human lactotransferrin, inhibits thrombin-induced platelet aggregation, secretion and thromboxane (TX) synthesis without interfering with phospholipase C (PLC) beta activation, since in previous work we have shown that Ca2+ mobilization and phosphorylation of the myosin light chain kinase (20 kDa) and pleckstrin (47 kDa) were normal. However, the inhibition of arachidonic acid-induced aggregation in the presence of KRDS is accompanied by normal TX synthesis suggesting that it does not interfere with the cyclooxygenase activity. To elucidate further the mechanisms of action of this peptide we tested its effect on U46619-induced platelet activation. KRDS inhibits U46619-induced platelet aggregation time- and dose-dependently without inhibiting the phosphorylation of pleckstrin. This suggests that the PLC pathway is not affected and that the inhibitory effect of KRDS is not due to and uncoupling of TXA2 from its receptor. In addition to the PLC pathway, protein tyrosine kinases play a major role in platelet signal transduction mechanisms. At least 7 tyrosine-phosphorylated proteins are detected upon stimulation of platelets by thrombin. KRDS strongly inhibits the tyrosine-phosphorylated substrates, in particular two 100-105 kDa substrates which are related to GP IIb/IIIa activation and platelet aggregation. The absence of TX synthesis observed in the presence of KRDS could be due to the inactivation of cPLA2 since the latter needs tyrosine phosphorylation to be activated, thus explaining the inhibitory action of KRDS on platelet functions.

The effects on platelet activation of two different tetrapeptides, KRDS present on human lactotransferrin and RGDS present on adhesive proteins such as human fibrinogen α-chain, were compared by a combination of morphological and functional techniques. Ultrastructural observations of α-thrombin stimulated platelets (0.05 U/ml), show strong platelet aggregation and full α-granule release. In the presence of RGDS (0.1-1 mM) aggregation was impaired but secretion was not blocked and platelets had released their α-granule contents. Platelets appeared uniformly degranulated with a dense central meshwork of microfilaments. In the presence of KRDS (0.5-1 mM), the platelets were activated with shape change and pseudopod formation. Aggregation was also impaired, but to a lesser extent since RGDS is active at a concentration as low as 0.1 mM, and, in contrast to RGDS, secretion was severely reduced. Electron microscopy showed that numerous α-granules were still scattered in the cytoplasmic matrix or often gathered in the centre of the platelet, but the majority of the open canalicular system cisternae remained clear. An immunoelectron microscopic study using immunogold and monospecific antibodies directed against fibrinogen and the a-granule membrane protein P-selectin (GMP 140) was performed. In the presence of RGDS, fibrinogen was released and P-selectin was translocated to the platelet surface; in contrast, in the presence of KRDS, fibrinogen remained localized in the α-granule, and the P-selectin associated with the a-granule. These observations were accompanied by some functional results: thrombin-induced platelet aggregation was inhibited by both peptides, and in contrast to RGDS, secretion was severely reduced in the presence of KRDS: serotonin release from dense granule was reduced by 73% compared to the control. These results show that these two tetrapeptides, in spite of some structural similarities, act differently in impairing platelet function. KRDS interfering with both the dense and α-granule release reaction may be a useful tool for a better understanding of the platelet secretion mechanism.

Interleukin-3 (IL-3), a cytokine known to be produced by activated T lymphocytes, mast cells, eosinophils and neutrophils, is a potent stimulator of normal haemopoiesis, particularly megakaryocytopoiesis. However, it remains unknown whether leukaemic megakaryoblasts can produce IL-3 and whether IL-3 is involved in the pathological process of megakaryoblastic leukaemia. In this study, several human leukaemia cell lines with or without megakaryocytic features, the DAMI, MEG-01, HEL, K562, HL-60 and U937, were chosen as the models. It was first demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) and indirect immunofluorescence assay that IL-3 was expressed in DAMI and MEG-01 cells, but not in other cell lines, although two erythroleukaemic cells, the HEL and K562, also possess some megakaryocytic features. Interestingly, the mRNA for IL-3 receptor was detected in nearly all the cell lines except K562 cells, suggesting that expression of IL-3 and its receptor may be dissociated in most of the cell lines and that co-expression of IL-3 and its receptor exists in megakaryoblastic cell lines, the DAMI and MEG-01. Of the cell lines which did not express IL-3 under unstimulated condition, only HEL cells were able to express IL-3 mRNA after treatment with PMA for 72 h. Furthermore, the proliferation of DAMI and MEG-01 cells could be enhanced in the presence of IL-3 and suppressed by the anti-IL-3 antibody and the IL-3 antisense oligodexyonucleotides (ODNs). These findings indicate that IL-3, as an autocrine growth factor, is involved in the growth of some megakaryocytic leukaemia cell lines.