Irene R. Katz

Platelet Factor 4: Production, Structure, and Physiologic and Immunologic Action

Platelet factor 4 (PF4) is a protein found in megakaryocytes and platelet α-granules (1, 2). Immunocytochemical studies show that it is present as well in mast cell granules (3) and on the endothelium of human umbilical veins, but not arteries (4). Early work on PF4 has been reviewed elsewhere (5–7). Human platelets contain about 18 ± 4 μg of PF4/109 (8). Thrombospondin, platelet-derived growth factor, and compounds derived from platelet basic protein such as β-thromboglobulin (β-TG) are found in platelet α-granules in addition to PF4. All are secreted when platelets are appropriately stimulated; for example, since thrombin is a strong stimulus, these compounds are present in much higher concentrations in serum than in plasma (e.g., 5334 vs 1.8 ng/ml for PF4 [9]). They are also secreted after contact of platelets with collagen in damaged blood vessels, for example. Production and Structure PF4 is synthesized by megakaryocytes (10), an ability that correlates with cytoplasmic maturity (2, 11). The PF4 is first packaged into vesicles and from there it is transferred to α-granules (2). Megakaryocytes express mRNA transcripts for PF4, but not for fibrinogen or albumin, which are taken up by megakaryocytes from plasma (12). Even blood platelets, which contain little mRNA, contain mRNA for PF4, whereas none is evident in human lymphocytes, cultured fibroblasts, and four types of malignant cells (13). Human PF4 is a 7.8-kDa protein that contains 70 amino acids, with two disulfide bonds, no tryptophan or methionine, two histidines, and a single tyrosine (Fig. 1). The position of its two disulfide bridges has been inferred by homology with the related compound β-TG (14). Its isoelectric point is 7.6 (15) and its extinction coefficient (1%, 280 μm) is reported to be 2.9 (3) or 5.4 (9).

Growth of SJL/J-derived transplantable reticulum cell sarcoma as related to its ability to induce T-cell proliferation in the host: III. Studies on thymectomized and congenitally athymic SJL mice

Abstract When SJL mice are irradiated and reconstituted with syngeneic bone marrow (XBM) they support growth of transplantable reticulum cell sarcoma to approximately 60% of that in normal mice. The ability to support RCS growth gradually improves with time after irradiation and reaches 90% of normal by 8–12 weeks. However, if the mice are thymectomized 4 weeks prior to treatment (Tx-XBM) they initially show 50% which increases to only 65% of growth in normal mice after 12 weeks. The ability of lymphoid cells from these mice to proliferate in vitro in response to irradiated RCS cells is normal 4 weeks after treatment in XBM, but remains in vivo possibly via their tendency to proliferate upon exposure to RCS.

Growth of SJL/J-derived RCS as related to its ability to induce T cell proliferation in the host. II. Negative influence of H-2d1.

Backcross SJL x (SJL x BALB/c)F1 and (SJL x BSVS)F1 mice were examined for their ability to support growth of transplantable SJL lymphoma (reticulum cell sarcoma (RCS). A marked linkage to H-2 was noted in that H-2s/d backcross mice failed to support tumor growth, while H-2s/s backcross mice showed approximately 70% of the growth seen in SJL mice, as judged by lymph node and spleen weights. Spleen cells obtained from backcross mice by splenectomy were examined for their ability to give proliferative responses to gamma-RCS cells, whereafter individual splenectomized mice were also examined for their ability to support lymphoma growth. Both properties showed a similar degree of linkage to H-2 and to each other, although there seemed to be a segregating non-H-2 BALB gene which also exerted an additional, less marked negative influence on the proliferative responses. It is suggested that the proliferative response in vivo may contribute to the lymphoma growth and that the presence of H-2d is inhibitory. (SJL x BSVS)F1 mice gave excellent proliferative responses and supported growth of RCS to approximately 80% of those of controls. These results confirm previous conclusions on the negative effect of H-21d in F1 hybrids on both phenomena.

Growth of SJL/J-derived transplantable reticulum cell sarcoma as related to its ability to induce T-cell proliferation in the host: IV. Effect of thymectomy on primary lymphoma incidence☆

Abstract The incidence of spontaneous primary lymphoma following adult thymectomy, irradiation, and bone marrow reconstitution was studied in SJL/J mice. A significantly higher incidence of generalized lymphoma was demonstrated in γ-irradiated and bone marrow-reconstituted (XBM) mice as compared to thymectomized, γ-irradiated, and bone marrow-reconstituted (ATx-XBM) mice. Including both localized and generalized lymphomas, ATx-XBM mice showed about half the incidence of XBM mice at 12 months of age. In comparison of XBM vs untreated controls, a much lower incidence of spleen involvement was noted in XBM mice than in untreated controls.

Reversal of Con A-induced suppression by a platelet-derived factor which binds to activated suppressor T cells

A platelet-derived factor found in serum as well as in platelet releasate prepared either with calcium ionophore or with thrombin was shown to reverse Con A-induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) in vivo in (CB6)F1 mice. In addition, as shown previously, lymphoma cell-induced suppression in SJL mice was similarly reversed. The factor could be injected prior to Con A on the day before SRBC injection, or on the same day as antigen with comparable results. It also enhanced PFC responses in the absence of Con A. Suppressor cell induction by Con A in vivo, as demonstrated by assay on PFC responses of normal spleen cells in vitro, was abrogated by simultaneous injection of the platelet factor. Cells from mouse spleen and lymph node, but not from thymus could absorb the factor from human serum at 4 degrees C. The phenotype of the relevant spleen cells was L3T4-, Ly1-, Ly2+, Thy1+, Ly22+, Qa1+, Qa4+, Qa5+, and Ly6.IE+. These results suggest that this factor binds to activated peripheral T cells of the suppressor cell phenotype.

Nonspecific immune modulating effects of ascites fluid and hyperimmune sera in vivo

To determine whether the appearance of interferon (IFN) and the modulation of humoral responses observed following injection of irradiated tumor cells were mediated by suppressor cells, the effects of in vivo injection of I-Js specific antibodies were studied. We found that anti-I-J-containing, as well as normal ascites fluids, obtained after repeated ip injection of complete Freunds adjuvant, contain a factor which (a) induces the appearance of serum IFN, (b) enhances the response to SRBC, and (c) suppresses the response to TNP-Ficoll, when injected 1 day before antigen. This effect is not immunologically specific, is probably not caused by intact Ig, and does not appear to be mediated by T cells. Although the nature of the factor(s) responsible for the observed results has not been fully clarified, we report these findings now as a cautionary note for the interpretation of studies where in vivo injection of unfractionated ascites fluids containing monoclonal antibodies are used.