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Thrombin, a multifunctional protein, has been found to be involved in cellular mitogenesis, tumor growth, and metastasis, in addition to its well known effects on the initiation of platelet aggregation and secretion and the conversion of fibrinogen to fibrin to form blood clots. These properties of thrombin rely on its action as a serine protease, which cleaves the N-terminal region of a 7-transmembrane G protein receptor (protease-activated receptor, PAR-1), thus exposing a tethered end hexapeptide sequence capable of activating its receptor. Little is known about its effect on genes that regulate the cell cycle. This study was undertaken to investigate the possible mechanisms by which thrombin regulates tumor cell growth in several tumor cell lines: human CHRF megakaryocyte, DU145 prostate, MDAMB231 and MCF7 breast, U3A fibrosarcoma, and 2 murine fibroblast cell lines, MEFp53(-/-) and CD STAT(-/-). We have found that thrombin under the conditions of culture employed inhibits cell growth by both up-regulation of p21(waf/cip1) and induction of caspases via its PAR-1 receptor. The increased expression of p21(waf/cip1) by thrombin was p53 independent, STAT1 dependent, and protein synthesis independent. This was associated with tyrosine phosphorylation of JAK2 and STAT1, and nuclear translocation of STAT1. Induction of apoptosis is also PAR-1-specific, STAT1-dependent, and associated with up-regulation of caspases 1, 2, and 3. Our study establishes, for the first time, a link between PAR-1 receptor activation with the STAT signal pathway, which leads to cell cycle control and apoptosis. This observation broadens our understanding of the mechanism of PAR-1 activation and its effect on cell growth, and could possibly lead to therapeutic approaches for the treatment of cancer.

Cellular-mediated immunity was studied in autoimmune thrombocytopenic purpura (ATP) patients by investigating leukocyte migration inhibition (LMI) following the interaction of normal platelets with patients' lymphocytes. When normal platelets were incubated with leukocyte buffy coats of ATP patients, the migration index (MI) was significantly impaired compared to buffy coats from normal subjects, employing 4 different concentrations of platelets. At the highest platelet concentration (10(9)/ml), MI was 0.87 +/- 0.04 (SEM) for ATP lymphocytes compared to 1.05 +/- 0.05 (p less than 0.01) for normal lymphocytes. Nine of 21 patients had an MI less than 0.80, whereas all control subjects had MIs greater than 0.85. Similar results were obtained at 2 different platelet membrane concentrations. At 500 micrograms/ml, the MI for ATP lymphocytes was 0.74 +/- 0.04, compared to 0.98 +/- 0.08 (p less than 0.01) for normal lymphocytes (12 experiments). An inverse relationship was noted between platelet count and lymphokine production in ATP patients (r = 0.815, p less than 0.001, 10 experiments). Transfer factor from an ATP patient in remission converted an abnormal LMI response of 0.68 +/- 0.04 from a patient with severe thrombocytopenia to 0.84 +/- 0.07 (p less than 0.005, 8 experiments). Similar results were obtained with transfer factor from 2 other patients in remission. Transfer factor from a patient with severe thrombocytopenia converted a normal response of 1.04 +/- 0.05 of normal subjects to a lower response of 0.88 +/- 0.04 (p less than 0.03, 12 experiments). Thus, lymphocytes of ATP patients are primed to recognize and be perturbed by normal platelets, whereas normal lymphocytes are not. This indicates specificity of the antigen- lymphocyte reaction in ATP patients. Transfer factor is capable of modulating this response in vitro.

The autologous mixed lymphocyte reaction (auto-MLR) measures the ability of non-T cells to stimulate autologous T cells to proliferate in tissue culture. The auto-MLR was studied in 11 patients with autoimmune thrombocytopenic purpura (ATP). Seven patients had decreased auto-MLR, which averaged 4440 +/- 3364 cpm (SEM) compared to 15,360 +/- 6905 cpm for simultaneously studied controls. The average of the ratios of cpm incorporated by patients/cpm incorporated by control subjects was 0.20 +/- 0.06 (p less than 0.01). Serum from all 7 patients with low auto-MLR decreased the auto-MLR of normal subjects by an average of 56% +/- 8.5 (p less than 0.001). Preliminary results indicate that the inhibitory effect was mediated by a component of the IgG immunoglobulin fraction of serum. Sera from normal persons and from ATP patients with normal or high auto-MLR did not affect the auto-MLR of normal subjects. It was further shown that non-T cells from 3 of 5 patients with decreased auto-MLR failed to stimulate allogeneic T cells normally. It is concluded that many patients with ATP have decreased auto-MLR apparently due to the presence of a serum blocking factor and, perhaps, a defective stimulatory capacity of non-T-cells.

Adult autoimmune throbocytopenic purpura (ATP) is a platelet disorder that develops in certain individuals with a genetic as well as sex (female) predisposition following an environment event (?viral). This results in the production of an IgG antiplatelet antibody capable of reacting with the host's platelets, as well as crossing the placenta. This leads to the rapid clearance and destruction of opsonized platelets by the reticuloendothelial system, particularly the spleen, by greater than tenfold the normal rate. Bound platelet IgG correlates with disease severity, whereas serum antiplatelet IgG does not. It has not been rigorously established whether bound platelet IgG is directed against a platelet antigen or represents an immune complex bound to the platelet Fc receptor. Nevertheless, several lines of evidence suggest that antiplatelet IgG binds directly to a platelet antigen(s). Megakaryocyte number, volume, and mass are increased commensurate with increased platelet turnover. Platelets of increased size, megathrombocytes, are noted on peripheral smear or via platelet volume distribution analysis. Megathrombocyte number is proporationate to megakarocyte number and to platelet turnover. Megathrombocyte diameter is inversely proportional to platelet survival. Antiplatelet antibody is also associated with qualitative platelet functional defects, which are indistinguishable from those noted with thrombopathia (i.e., apparent platelet release defect). Antibody-induced functional defects are probably more common than quantitative thrombocytopenic defects and may represent a significant portion of those women with the “easy bruising” syndrome and normal platelet count. Adults who develop ATP generally develop the chronic variety, which remains permanently with the patient. Treatment should be directed towards maintaining the patient free of purpura, not restoring the platelet count to normal. This can generally be accomplished with a platelet count of < 40,000/cu mm with patients having this disorder. Approximately 50% of patients respond to steroids by a significant elevation of platelet count and improvement of purpura. However, cessation of therapy results in eventual relapse if the disease is of the chronic variety. Splenectomy is successful in approximately 65–75% of patients, resulting in a restoration of the platelet count to normal or safe levels by removing a major source of platelet destruction as well as antibody production; platelet survival improves. At least 50% of patients “in remission” following steroids or splenectomy generally have a compensated thrombocytolytic state in which increased platelet production keeps up with increased platelet destruction. Antiplatelet IgG can often be found in the serum of these patients. Patients refractory to steroids and/or splenectomy present with a serious therapeutic problem. Immunosuppressive therapy is effective in approximately one-third of refractory patients, but often relapses occur, requiring maintenance therapy with potentially mutagenic drugs...