Inhibition of platelet-derived growth factor-induced mitogenesis and tyrosine kinase activity in cultured bone marrow fibroblasts by tyrphostins.

Tyrphostins, which block protein tyrosine kinase activity, were studied for their inhibitory action on platelet-derived growth factor (PDGF)-induced proliferation of human bone marrow fibroblasts. Of the seven tryphostins examined, tyrphostin AG370 was found to be the most potent blocker against PDGF-induced mitogenesis (IC50 = 20 microM). This PTK blocker also blocks mitogenesis induced by epidermal growth factor (IC50 = 50 microM) and human serum (IC50 = 50 microM), but with lower efficacy. In digitonin-permeabilized fibroblasts as well as in intact fibroblasts, tyrphostin AG370 inhibits PDGF receptor autophosphorylation and the tyrosine phosphorylation of intracellular protein substrates (pp120, pp85, and pp75) which coprecipitate with the PDGF receptor. In comparison to AG370, AG18, a potent EGF receptor blocker, was less efficient in inhibiting PDGF-induced proliferation of fibroblasts and phosphorylation of the intracellular protein substrates. Under the conditions in which AG370 inhibits PDGF-induced mitogenesis and phosphorylation, it does not affect [125I]PDGF internalization and enhance [125I]PDGF binding. These findings suggest that AG370, which is an indole tyrphostin, may serve as a model for developing analogues with a therapeutic potential for treatment of diseases which involve abnormal cellular proliferation induced by PDGF.

Increased intraplatelet levels of platelet-derived growth factor and transforming growth factor-beta in patients with myelofibrosis with myeloid metaplasia.

Platelet-derived growth factor (PDGF) is thought to play some role in the genesis of fibrosis associated with myeloproliferative disorders. In addition, transforming growth factor-beta (TGF-beta) has been confirmed to promote fibrotic process. Both PDGF and TGF-beta have been shown to cooperate with epidermal growth factor (EGF) in regulating the growth of human marrow fibroblasts. All three are contained in platelet alpha-granules. We report the results of a study in patients with myelofibrosis with myeloid metaplasia (MMM). We evaluated PDGF, TGF-beta and EGF-like activities in circulating platelets from patients compared to healthy subjects. In contrast to EGF-like intraplatelet levels which were similar in patients and in normal donors (1-4 ng/10(9) platelets), we found constantly higher values for both PDGF and TGF-beta in MMM patients. In both radioimmunoassay (RIA) and assay for mitogenic activity on human bone marrow fibroblasts, PDGF levels were increased on the average 2-3.5-fold over the levels found in normal donors (P less than 0.01 and P less than 0.001, respectively). PDGF serum levels in patients were consistent with those found in platelets. In platelet-poor plasma (PPP), PDGF concentrations were undetectable or congruent to 2 ng/ml in patients and in control donors as well. The total TGF-beta activity in platelet lysates, determined using a competitive radioreceptor binding assay on Swiss 3T3 mouse cells and an inhibition growth assay on CCL64 cells, was found 2-3-fold increased in patients with MMM as compared to control subjects (P less than 0.003). These results emphasize that, not only PDGF, but also TGF-beta are implicated in the myelofibrosis with myeloid metaplasia.

Platelet-derived growth factor (PDGF) in type 1 diabetes mellitus.

In 10 patients with type 1 diabetes mellitus, platelet-derived growth factor (PDGF) was assessed using an in vitro assay based on the stimulation of DNA synthesis in the 3T3 mouse fibroblast cell line. beta-Thromboglobulin (beta-TG) was used as an index for platelet alpha-granules content. Platelet beta-TG content and PDGF were markedly decreased in diabetic patients while plasma beta-TG was increased as compared with control subjects. In diabetic patients, a significant negative correlation was found between plasma beta-TG level and beta-TG total platelet content, associated with a significant positive correlation between platelet beta-TG content and PDGF. These results suggest that PDGF release might be increased in diabetic subjects. This may account in part for the cell proliferation observed in diabetic angiopathy.

Collagen-induced binding to human platelets of platelet-derived growth factor leading to inhibition of P43 and P20 phosphorylation.

Platelet-derived growth factor (PDGF) is known to inhibit collagen-induced platelet aggregation. Collagen-induced binding of 125I-PDGF to human washed platelets was therefore investigated. It was found 1) to be time-dependent, reaching a plateau at 20 degrees C after 30 min, 2) collagen concentration-dependent, 3) specifically inhibited by unlabeled PDGF, and 4) saturable. Scatchard plot analysis showed a single class of sites with 3000 +/- 450 molecules bound/cell and an apparent KD of 1.2 +/- 0.2 10(-8) M. The effects of PDGF on collagen-induced phosphoinositide breakdown and protein phosphorylation were also investigated. At 50 ng/ml PDGF, a concentration which completely inhibited collagen-induced aggregation, the breakdown of [32P]phosphatidylinositol 4,5-biphosphate (PIP2) and [32P]phosphatidylinositol 4-phosphate (PIP) was observed, but the subsequent replenishment of [32P]PIP2 was inhibited. The same PDGF concentration totally inhibited collagen-induced phosphatidic acid formation. PDGF also completely prevented phosphorylation of P43 and P20, as a result of protein kinase C activation consecutive to phosphoinositide metabolism. These results suggest that (i) a specific PDGF receptor can be induced by collagen, and (ii) PDGF can effect the early events of collagen-induced platelet activation by inhibiting PIP2 resynthesis and P43 and P20 phosphorylation. It is concluded that PDGF might be involved in a negative feed-back control of platelet activation.

Transforming growth factor (TGF beta) decreases the proliferation of human bone marrow fibroblasts by inhibiting the platelet-derived growth factor (PDGF) binding.

Human bone marrow fibroblasts were cultivated and characterized by immunofluorescent staining and electron microscopy. Their interactions with PDGF and TGF beta were studied. While a positive intracellular antifibronectin staining was observed, the cultured cells were not labeled with specific antibodies toward factor VIII von Willebrand factor (F VIII/vWF), desmin, and macrophage antigen. Moreover, electron microscopy excluded the presence of endothelial cells by the absence of Weibel-Palade bodies. The binding of pure human PDGF to the cultured bone marrow fibroblasts was investigated. Addition of an excess of unlabeled PDGF decreased the binding to 75 and 80%, which means that the nonspecific binding represented 20-25% of total binding, whereas epidermal growth factor (EGF) had no effect. Two classes of sites were detected by Scatchard analysis with respectively 21,000 and 37,000 sites per cell, with a KD of 0.3 x 10(-10) M and KD of 0.5 x 10(-9) M. The stimulation of DNA synthesis by PDGF was quantified by [3H]thymidine incorporation. When PDGF was added alone at a concentration of 15 ng/ml, it induced a maximal DNA synthesis of 400%, which increased up to 900%, in the presence of platelet-poor plasma (PPP). On the other hand, PDGF-induced fibroblast proliferation was inhibited in a dose-dependent manner by TGF beta. This inhibition was related to a significantly decreased binding of 125I-labeled PDGF observed in the presence of TGF beta. Our results suggested that PDGF and TGF beta could modulate the growth of bone marrow fibroblasts.

Megakaryocytes and myelofibrosis in gray platelet syndrome.

Studies have been carried out with cultured human megakaryocytes derived from Gray Platelet Syndrome blood and have demonstrated that MK-DGF and MK-F4 are present in the early stages of megakaryocyte maturation (day 5-6) but disappear in the late phase (day 9-11). In Gray platelets total mitogenic activity, PDGF and PF4 amounts were reduced while beta TG and PF4 plasma levels were slightly increased. These findings indicate a possible relationship between the loss of alpha granule contents from the megakaryocytes in the marrow and the myelofibrosis found in Gray platelet disorder and possibly associated in other diseases with platelet or megakaryocyte abnormalities.

Platelet acquired defect in PDGF and beta thromboglobulin content in hairy cell leukaemia: improvement after interferon therapy.

To investigate the platelet contribution to the development of myelofibrosis in hairy cell leukaemia (HCL), we have studied two platelet alpha granule components in 15 patients with HCL before chemotherapy: mitogenic activity was measured by 3H thymidine incorporation in BALB/C 3T3 cells and beta thromboglobulin (beta TG) assayed by radioimmunoassay (RIA). Platelet mitogenic activity and beta TG content were significantly decreased in the patients as compared to the control subjects. The nine patients who were treated with recombinant human interferon (IFN alpha A) were restudied after 4 months of therapy. The levels of both mitogenic activity and beta TG platelet content were significantly increased after IFN alpha-treatment with a complete response in five of the nine treated patients, a partial response in two and no response in the two others. HCL seemed therefore to be responsible for an acquired platelet alpha granule defect. As in the grey platelet syndrome a relationship between this abnormal platelet granule storage and the development of myelofibrosis is suggested in HCL.

PDGF modifies phosphoinositide metabolism and inhibits aggregation and release in human platelets.

While platelet derived growth factor (PDGF) did not induce any platelet aggregation nor secretion, it modified the polyphosphoinositide metabolism of human platelets prelabeled with 32P-orthophosphate. We found a decrease of 32P associated with phosphatidylinositol 4,5 bisphosphate after 3 min, with parallel increase of 32P-phosphatidylinositol 4 phosphate and 32P-phosphatidylinositol using 100 ng/ml of PDGF. This modification was PDGF concentration dependent. PDGF inhibited thrombin and collagen induced platelet aggregation and 14C-serotonin release in a dose dependent manner, but was without effect when arachidonic acid was used. These results suggest that PDGF (i) stimulated the hydrolysis of polyphosphoinositides (ii) and could exert a negative feedback control on platelet activation induced by thrombin or collagen.

Human platelet-tumor cell interactions vary with the tumor cell lines.

Platelets may promote the development of metastasis, and tumor cells that aggregate platelets are believed to be more malignant. We studied three different human mammary carcinoma cell lines, which had different interactions with human platelet-rich plasma (PRP). The MCF-7 and the T47-D cell lines induced an adenosine diphosphate (ADP)-mediated platelet aggregation. The third cell line, MDA-MB 231 did not induce any platelet aggregation. On the contrary, this cell line inhibited ADP- and arachidonic acid-induced platelet aggregation. This inhibiting activity is mainly adenosine-mediated. The mechanism by which platelets may contribute to the dissemination of cancer could be related to platelet growth factors. MCF-7 and T47-D cell lines induced a release of platelet-derived growth factor (PDGF). On the contrary, the MDA-MB 231 cell line did not induce any platelet release. The role of these platelet growth factors in tumor cell growth is discussed.

Abnormality of glycoprotein Ib in two cases of "pseudo"-von Willebrand's disease.

Two unrelated patients with "pseudo" ("platelet-type")-von Willebrand's disease (vWD) are described demonstrating thrombocytopenia with a prolonged bleeding time, ristocetin-induced platelet aggregation at low stimulus concentrations, decreased levels of ristocetin-cofactor activity (vWF:RCo), slight cryoprecipitate-induced platelet aggregation in the absence of ristocetin, and a lack of the high molecular weight factor VIII-von Willebrand factor (FVIII/vWF) multimers in plasma. Isolated washed patient platelets bound more FVIII/vWF at high (1 and 0.75 mg/ml) and low (0.5 and 0.25 mg/ml) ristocetin concentrations than control platelets. Fresh or paraformaldehyde-fixed washed platelets from these patients also bound more specific monoclonal antibody to glycoprotein Ib (25,000 binding sites per cell) than normal platelets (15,000 +/- 3,300 binding sites per cell). Results obtained in control patients with thrombocytopenia and increased platelet size (May-Hegglin anomaly and vWD type IIB) excluded a nonspecific increase of glycoprotein Ib in the platelets of the patients with pseudo-vWD. These data indicate that in pseudo-vWD, the primary abnormality lies in the platelet and is related to a quantitative and/or qualitative anomaly of platelet membrane glycoprotein Ib.

The domain of platelet glycoprotein I as recognized by various antibodies, both monoclonal and polyspecific.

Platelet membrane glycoprotein Ib plays a major role in the binding of factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium. We have used polyspecific and monoclonal antibodies to glycoprotein Ib and have demonstrated that both antibodies were directed to glycoprotein Is, a soluble fragment of glycoprotein Ib. By showing an inhibition of the binding of factor VIII/von Willebrand factor to control platelets in presence of the antibodies, it can be concluded that glycoprotein Is is involved in these binding sites.

Correlation of enzymatic activity and anticoagulant properties of phospholipase A2.

Some highly purified phospholipases A from the venom of viperidae, crotalidae and elapidae were found to hve anticoagulant properties. All phospholipases which exhibited anticoagulant properties are characterized by a high isoelectric point, but not all strongly basic phospholipases are anticoagulant. Anticoagulant phospholipases hydrolyse highly packed monomolecular films of phospholipids without any lag time while non-anticoagulant phospholipases present considerable induction times indicative of a low penetrating power. When the ester linkages in the procoagulant lipids were replaced by the non-hydrolysable ether bonds, the mixture retained its clotting ability even in the presence of phospholipases, thus suggesting that anticoagulant phospholipases prevent clot formation by hydrolysis of phospholipids. This was confirmed by chemical modification of phospholipases, viz. alkylation of the active-centre histidine with 1-bromo-octan-2-one. This modification yielded proteins which had lost their anticoagulant properties but which retained a high affinity for phospholipids.