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In vitro megakaryocyte colony formation was studied in 25 patients with chronic idiopathic thrombocytopenic purpura (ITP), and compared to 16 controls. A significantly lower number of megakaryocyte progenitors (CFU-MK) was observed in patients (10.8 +/- 12.0 CFU-MK per 10(5) bone-marrow non-adherent mononuclear cells (NAMC), compared to controls (22.5 +/- 15.4 CFU-MK); P < 0.002. Only six patients had a normal number of CFU-MK. The number of CFU-MK was not correlated to initial platelet count (P = 0.35). These findings indicate that in vitro CFU-MK could be of interest in the diagnosis of chronic ITP, and also could be predictive for chronicity in patients with acute ITP.

Seventeen patients with primary thrombocythemia (PT) were evaluated for in vitro bone marrow megakaryocyte progenitors (CFU-MK) using a plasma clot system. The aim of this study was to find out whether spontaneous growth of CFU-MK could be used in the diagnosis of PT. The number of CFU-MK was normal in 7 patients and reduced in 10 patients. In the absence of stimulating factor, CFU-MK grew spontaneously in 12 patients, while in 5 patients no spontaneous CFU-MK were observed. The mean plasma level of platelet factor 4 (PF4) was significantly higher (p less than 0.05) in patients without spontaneous CFU-MK (59.8 +/- 59.6 IU/ml; mean +/- SD) compared to patients with (18.1 +/- 20.7 UI/ml). The mean plasma level of beta-thromboglobulin did not differ between patients with or without spontaneous CFU-MK. The beta-thromboglobulin/PF4 ratio was significantly higher (p less than 0.01) in patients with spontaneous CFU-MK (9.9 +/- 7.1) compared to patients without (3.1 +/- 1.4). These results suggest that PF4 could inhibit in vitro spontaneous growth of CFU-MK.

In vitro bone-marrow megakaryocyte colony formation was studied in 10 patients with HIV-associated thrombocytopenia to investigate the mechanism of thrombocytopenia. Increased colony formation was observed in 3 patients and decreased growth in 7 patients. No relationship was noted between the growth potential of megakaryocyte progenitors and platelet count, number of CD4+ celts, platelet response to azidothymidine, and platelet count 7 days after culture. In all patients, megakaryocyte morphology was abnormal: blebbing of the membrane and abnormal chromatin with separated lobes of nuclei. Further studies are needed to determine if growth potential of megakaryocyte progenitors is useful in understanding the mechanism of thrombocytopenia in HIV-infected individuals.

In vitro megakaryocyte colony formation from the bone marrow of patients with acute idiopathic thrombocytopenic purpura (ITP) or chronic ITP was compared using a plasma clot system. The number of megakaryocyte colony-forming units (CFU-Meg) was significantly higher (p less than 0.05) in acute ITP compared to chronic ITP (54.3 +/- 68.4 vs. 12.9 +/- 15.3/10(5) nonadherent mononuclear cells, mean +/- SD), and significantly lower (p less than 0.05) in chronic ITP compared to controls (12.9 +/- 15.3 vs. 22.8 +/- 15.9). A significant correlation was observed between platelet recovery 7 and 30 days after culture, and the number of CFU-Meg (r = 0.49 and 0.45, respectively, p less than 0.05). An inverse correlation was observed between platelet count at the time of culture and the number of Megs per colony (r = -0.48, p less than 0.05). These results indicated a difference between acute and chronic ITP in the ability to promote in vitro Meg colony formation and may suppose a different immune mechanism for thrombocytopenia in these two disorders.

Seven tetramethylrhodamine B isothiocyanate- (TRITC) labeled lectins: lens culinaris (LCH), ulex europeus-1 (UEA-1), lycopersicon esculentum (LEA), wheat germ agglutinin (WGA), dolichos biflorus (DBA), soybean agglutinin (SBA) and erythrina cristagalli (ECA) were applied on cultured human megakaryocytes (Megs) detected by immunofluorescence. All stages of Megs (from lymphocyte-like Megs to mature Megs) and platelets were labeled by LCH, LEA, UEA-1 and WGA. ECA binds to platelets but only to some Megs. DBA did not bind to platelets but did bind to some Megs, irrespective of stage. SBA binds to all stages of Megs, but did not bind to platelets. These results indicate the presence of mannose, glucose (LCH), sialic acid (WGA), and glucosamine (UEA-1, LEA, WGA) on the surface of all cells of the Meg lineage, a variable presence of galactosamine (DBA, SBA, ECA), and a discrepancy in the presence of some galactosamine compounds between platelets and Megs (DBA, SBA).

The effect of highly purified human beta-thromboglobulin (beta TG), a glycoprotein present in platelet alpha granules, was tested on human bone marrow in vitro megakaryocyte (MK) colony formation. A concentration of 5 micrograms/ml of beta TG induced a 50% reduction in the number of MK colonies, and concentrations of 10 and 20 micrograms/ml completely inhibited MK growth. This inhibition was of importance for MKs because a higher concentration of beta TG (10 micrograms/ml) was needed to obtain a nonsignificant decrease in erythroblastic progenitors (erythroid burst-forming units, BFU-E), and no inhibition of granulocyte-macrophage colony-forming units (CFU-GM) was observed. beta TG acts mainly on maturation of MKs. These results indicate that beta TG could play a role in the physiological regulation of platelet production by MKs.

Platelet factor 4 (PF4) is a multifunctional protein specific to platelets, synthesized in megakaryocytes and stored in alpha granules. This report of our work shows that PF4 potently inhibits human megakaryocyte colony formation in vitro. Colony formation by megakaryocyte progenitor cells from normal bone marrows was studied using the plasma clot culture system and indirect immunoperoxidase staining. Nonadherent mononuclear cells were co-cultured with various concentrations (0 to 20 micrograms/mL) of highly purified human PF4. Statistically significant inhibition of three classes of megakaryocyte progenitor cells, the mixed colony forming unit-megakaryocytes (mCFU-MK), the burst forming unit-megakaryocytes (BFU-MK), and the colony forming unit-megakaryocytes (CFU-MK), was seen at a PF4 concentration of 2.5 micrograms/mL or greater. PF4 had no effect on erythroid (BFU-E) and granulocyte/macrophage (CFU-GM) colony formation except at high concentration (5 micrograms/mL for BFU-E and 10 micrograms/mL for CFU-GM). When a concentration of 5 micrograms/mL PF4 was added at various time points during marrow culture, a reduction of megakaryocyte colony formation also occurred. In the presence of PF4 2.5 micrograms or 5 micrograms/mL, the percentage of mature type of colonies was found to be decreased compared with cultures with no added PF4. These data demonstrate that PF4 inhibits both proliferation and maturation of megakaryocyte progenitor cells in vitro and suggest that PF4 may play a role in autoregulating human megakaryocytopoiesis.

Colony formation by megakaryocyte (MK) progenitors was studied in 36 normal individuals and in 26 patients with primary thrombocythemia (PT) using an improved plasma clot cloning system. MK colonies were identified by immunoperoxidase staining using a monoclonal antibody against human platelet glycoprotein IIb/IIIa complex. In normal individuals, a frequency of 194 +/- 23 MK colony-forming units (CFU-MK) per 5 X 10(5) bone marrow nonadherent mononuclear cells and 11 +/- 4 CFU-MK per 5 X 10(5) blood mononuclear cells was found. These CFU-MK grew as large (greater than 20 cells), mean (11-20 cells), small (3-10 cells), or mix-MK colonies (at least two MK) that comprised 15%, 23%, 62%, or 5% of all MK colonies in bone marrow and 0%, 8%, 92%, or 1% in peripheral blood, respectively. In PT, several abnormalities of MK colony formation were observed: 1) increased circulating CFU-MK numbers, 2) increased mix-MK colony formation, 3) spontaneous MK colony formation without phytohemagglutinin-stimulated leukocyte-conditioned medium and normal serum, 4) decreased proportion of larger MK colonies (greater than 11 cells) in PT bone marrow, and 5) failure of PT plasma or serum to stimulate MK colony formation by normal marrow cells in a normal fashion. These results indicate some of the characteristics of quantitative and qualitative abnormalities of in vitro megakaryocytopoiesis in PT.

Characteristics of circulating CFU-MK and the effect of serum and plasma on CFU-MK growth were studied in 14 patients with primary myelofibrosis (MF) using short- and long-term culture methods. The number of CFU-MK in short-term cultures was significantly increased in the non-splenectomized patient group (p less than 0.01). Without added PHA-LCM and normal serum, spontaneous colony formation was found in 9 out of 10 patients. In long-term blood cultures from 6 MF patients, 3 untreated patients formed confluent adherent layers and produced in suspension an equal number or an even greater number of nucleated cells, megakaryocytes and CFU-MK than those obtained in long-term bone marrow culture from normal individuals. 2 splenectomized patients showed neither an increased numbers of CFU-MK nor the capacity to develop an adherent layer. The serum and plasma of MF patients failed to stimulate megakaryocyte colony formation by normal bone marrow in a normal fashion. These findings indicate a megakaryocytopoietic abnormality, and a central role of the spleen in extramedullary haematopoiesis in MF.

An improved plasma clot culture method for CFU megakaryocytes (MK) has been developed with a higher plating efficiency and easier identification and enumeration of MK colonies by an indirect immunoperoxidase staining using a monoclonal antibody specific to the platelet glycoprotein IIb/IIIa complex. This technique has been used to study megakaryocytopoiesis in 20 normal individuals, 4 recently diagnosed patients with untreated primary thrombocythaemia (PT) and 2 patients with secondary thrombocytosis (ST). An increased number of MK colonies was evident in peripheral blood (mean 115 +/- 31 CFU-MK/5 X 10(5) seeded cells or 206 +/- 91/ml) and to a lesser extent in bone marrow (mean 188 +/- 26 CFU-MK/5 X 10(5) seeded cells or 696 +/- 103/ml) of PT patients as compared to controls (mean 11 +/- 4/5 X 10(5) seeded cells or 13 +/- 3/ml of peripheral blood and 153 +/- 15/5 X 10(5) seeded cells or 319 +/- 43/ml of bone marrow). There was a very obvious difference between PT patients and the others (controls plus ST patients) because CFU-MK growth with no added stimulus (PHA-LCM or PHA-LCM and normal serum) could be seen in PT patients only.

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) IIb-IIIa complex (AP2 and 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 mumol/L) inhibited the binding of AP2 and P2 on ADP (5 mumol/L)-stimulated platelets, but not on unstimulated platelets. However, the binding of P2 was considerably more inhibited than that of AP2 as judged by immunoperoxidase intensity. Radiolabeled AP2 binding 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 IIIa (SZ 21), GP IIb (SZ 22), and GP Ib (SZ 2) on ADP-stimulated human platelets. Inhibition of P2 binding by KRDS was also observed in a section of MKs isolated from human bone marrow and stimulated by 15 or 20 micron ADP. A lower concentration of ADP (5 or 10 mumol/L) failed to produce any inhibition of binding. This indicates that MKs may not be equally responsive to agonists as platelets. Moreover, P2 binding inhibition was observed in a larger (P less than .001) percentage of mature MKs (29%) as compared with younger, maturing MKs (11%). The observations suggested that a functional ability possessed by platelets, namely, agonist-induced exposure of the site of interaction of KRDS, may occur at a late stage of MK development.

Effects of recombinant human interferon alpha (HuIFN-alpha) on human megakaryocyte (CFU-MK) and fibroblast (CFU-F) colony-forming cell growth were studied. Concentration-dependent inhibition of both CFU-MK and CFU-F by HuIFN-alpha was demonstrated. Statistically significant suppression of both CFU-MK and CFU-F was seen at a HuIFN-alpha concentration of 1000 U/ml or greater. No significant difference was found between HuIFN-alpha treated cultures and controls for the distribution of CFU-MK types and for the size and cell morphology of CFU-F. When a concentration of 1000 u/ml HuIFN-alpha was added at varying time points during the marrow cultures, decreased numbers of megakaryocyte and fibroblast colonies only appeared at the early days of cultures. When bone marrow cells were incubated with HuIFN-alpha for different periods of time prior to initiation of cultures, a reduction of megakaryocyte colony formation also occurred. These studies demonstrate a suppressive effect of HuIFN-alpha on human CFU-MK and CFU-F growth. This effect seems to occur at the initial stages of CFU-MK and CFU-F development.