Therapeutic potential of Janus kinase 3 (JAK3) inhibitors.

Here we discuss the therapeutic potential of Janus kinase 3 (JAK3) inhibitors as a new class of immunomodulatory agents with immunosuppressive, anti-inflammatory, anti-allergic, anti-thrombotic and anti-leukemic properties. JAKs are abundantly expressed in primary leukemic cells from children with ALL (acute lymphoblastic leukemia) and are crucial for signals regulating apoptosis. Additional roles for JAK3 in mast cell-mediated immediate hypersensitivity reactions, autoimmune disorders and platelet function have recently been described. The preclinical studies on JAK3 inhibitors revealed their clinical potential as anti-leukemic agents with anti-thrombotic, anti-allergic and immunosuppressive properties. Results from multiple preclinical experimental model systems of autoimmune diabetes, pancreatic islet transplantation, solid organ transplantation, allergy, thrombosis and bone marrow transplantation are discussed in the context of the clinical need for new immunomodulatory agents with such properties.

Role of a JAK3-dependent biochemical signaling pathway in platelet activation and aggregation.

Here we provide experimental evidence that identifies JAK3 as one of the regulators of platelet function. Treatment of platelets with thrombin induced tyrosine phosphorylation of the JAK3 target substrates STAT1 and STAT3. Platelets from JAK3-deficient mice displayed a decrease in tyrosine phosphorylation of STAT1 and STAT3. In accordance with these data, pretreatment of human platelets with the JAK3 inhibitor WHI-P131 markedly decreased the base-line enzymatic activity of constitutively active JAK3 and abolished the thrombin-induced tyrosine phosphorylation of STAT1 and STAT3. Following thrombin stimulation, WHI-P131-treated platelets did not undergo shape changes indicative of activation such as pseudopod formation. WHI-P131 inhibited thrombin-induced degranulation/serotonin release as well as platelet aggregation. Highly effective platelet inhibitory plasma concentrations of WHI-P131 were achieved in mice without toxicity. WHI-P131 prolonged the bleeding time of mice in a dose-dependent manner and improved event-free survival in a mouse model of thromboplastin-induced generalized and invariably fatal thromboembolism. To our knowledge, WHI-P131 is the first anti-thrombotic agent that prevents platelet aggregation by inhibiting JAK3.

Platelets and DAMI megakaryocytes possess beta-secretase-like activity.

We report here the discovery of two novel human platelet and megakaryocytic DAMI cell enzymes that have beta-secretase-like activity. These activities could potentially effect cleavage of the amyloid precursor protein (APP) at the beta-amyloid peptide N-terminus, by an EC 3.4.24.15-like metalloprotease, and the N terminus-1 position, by a serine protease. Thus both enzymes may generate the amyloidogenic beta-peptide. Studies of intact and Triton X-100-lysed DAMI cells, as well as intact versus subcellular fractions of platelets, demonstrate the presence of these proteolytic activities. The resting platelet has (1) a surface serine protease, demonstrated by its ability to cleave a beta-secretase substrate and by its inhibitor sensitivity; and (2) a metalloprotease, recognized by an antibody to EC 3.4.24.15, which resides intracellularly in the alpha-granule membrane, is translocated to the surface on activation, and shows beta-secretase-like activity by cleaving the same substrate. This metalloprotease can also cleave recombinant APP to a potentially amyloidogenic fragment. Surface metalloprotease was identified in DAMI cells by flow cytometry and Western blotting with a specific anti-EC 3.4.24.15 monoclonal antibody, while activity was identified by using two beta-secretase substrates. This article is the first to document two previously unknown endoproteinases with beta-secretase-like activity in platelets and DAMI cells. These proteases are capable of effecting cleavage of APP and could therefore contribute to Abeta deposition in the cerebrovasculature.

Moderate and advanced Alzheimer's patients exhibit platelet activation differences.

We previously reported that platelets from advanced sporadic Alzheimer's disease (AD) patients exhibit two defects: first, an aberrant signal transduction presenting as a thrombin-induced hyperacidification, which is more severe for donors with the apolipoprotein E4 allele (apoE4), and second, an AD-specific Amyloid Precursor Protein (APP) processing defect that presents as retention of APP on the activated platelets' surface and in independent of the apo E allele. This retention of membrane APP correlates with decreased release of soluble APP. To determine at what stage in the disease progression these defects appear, we performed signal transduction and secretion studies on moderate AD patients. Thrombin-activated platelets from these patients do not exhibit either hyperacidification or APP retention; their APP processing and secretion are normal by Western blotting, suggesting that the two platelet defects appear in the advanced stages of AD.