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.

Blood brain barrier endothelial cells express candidate amyloid precursor protein-cleaving secretases.

Proteolytic cleavage of the amyloid precursor protein (A beta PP) results in the generation of the amyloidogenic fragment known as amyloid beta peptide (A beta). Deposition of A beta in the brain parenchyma and cerebrovasculature is a feature of Alzheimer's disease (AD). To date, the process whereby A beta is generated and deposited remains unclear. We have previously established that activated platelets from AD patients retain more A beta PP on their surface than control platelets. We report here that an endothelial cell-derived enzyme can cleave this surface platelet A beta PP. Human blood brain barrier endothelial cells from brains of AD patients were assayed for potential A beta PP-cleaving enzymes using synthetic peptide substrates encompassing the A beta N-terminus cleavage site. A protease activity capable of cleaving A beta PP on the surface of AD platelets was noted. The A beta PP cleavage is partially inhibited by EDTA, by ZincOV, as well as by a specific inhibitor of the Zn metalloprotease E.C.3.4.24.15. Furthermore, the protease is recognized by an antibody directed against it, using immunohistochemistry, Western blot analysis and flow cytometry. The protease is not secreted, but rather resides intracellularly as well as on the surface of the endothelial cells. The data suggest that E.C.3.4.24.15 synthesized by brain endothelial cells may process the platelet-derived A beta PP, yielding fragments which could contribute to cerebrovascular A beta deposits.

Brain endothelial cell enzymes cleave platelet-retained amyloid precursor protein.

We have previously demonstrated that thrombin-activated platelets from patients with advanced Alzheimer's disease (AD) retain significantly more surface membrane-bound amyloid precursor protein (mAPP) than platelets from non-demented age-matched individuals (AM). We have studied interactions between these platelets and the cerebrovascular endothelium to which activated platelets adhere in a model system, investigating their involvement in the formation of amyloid beta peptide (Abeta) deposits in AD patients. We report here that there appear to be alpha and beta secretase-like activities in primary human blood brain barrier endothelial cell (BEC) cultures from both AD patients and AM control subjects (AD-BEC and AM-BEC, respectively) as well as a gamma secretase-like activity that appears only in AD-BEC. No such activities were observed in human umbilical vein endothelial cells (HUVECs). Furthermore, there is more penetration of the platelet-released products platelet factor 4 and soluble APP through the BEC layer grown from AD patients than that grown from AM individuals, whereas none penetrate through a HUVEC layer. Thus the interaction between platelets, the APP they have retained or released, and cerebral vascular endothelial cells may be at least partially responsible for amyloidogenic deposits around the cerebral vasculature of AD patients.

Stimulus responses and amyloid precursor protein processing in DAMI megakaryocytes.

Platelets, when released as anuclear cells by their precursor megakaryocytes, already carry soluble proteolytic fragments of the amyloid precursor protein (APP) within their alpha-granules and intact APP in the alpha-granule membranes. In response to activation signals elicited by physiologic stimuli such as thrombin, platelets release their granules' soluble contents and translocate granule membrane-bound proteins to the plasma membrane. Because platelets carry >90% of the circulation's APP, activated platelets have been implicated as origins of the beta-amyloid peptide fragment of APP (A beta), whose deposition in the cerebrovasculature is characteristic of Alzheimer's disease. We have therefore studied the APP contents and proteolytic processing in resting DAMI human megakaryocytic cells, along with the consequences of the activation of these cells by thrombin, comparing the results in each case to those with human platelets. Resting and PMA-differentiated DAMI cell contents were examined by Western blotting, immunoprecipitation, or metabolic labeling with sulfur 35-labeled methionine during culture, while plasma membrane-bound APP was evaluated by flow cytometry. Activation was followed by changes in cytoplasmic calcium concentration ((Ca++)in) and in membrane potential. Like platelets, DAMI cells exhibited a thrombin dose-dependent delta(Ca++)in, and membrane potential change; in contrast to the surface of a platelet, the surface of an agranular resting DAMI cell expresses granule-membrane proteins (APP and CD63) that appear on platelets only after activation. DAMI cell culture with 35S-labeled methionine confirmed that megakaryocytes synthesize large amounts of APP, of slightly higher molecular weight, and degrade their APP extensively before platelets are formed.

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.

Activated Alzheimer disease platelets retain more beta amyloid precursor protein.

Upon activation, platelet alpha-granules' soluble contents are secreted and membrane-bound contents are translocated to the plasma membrane. Membrane-bound proteins include the beta-amyloid precursor protein (APP) from which the beta-amyloid (A beta) deposits found surrounding the cerebrovasculature of patients with Alzheimer's Disease (AD) may originate. We show here that activated platelets from AD patients exhibit less APP processing, retain more of the protein on their surface, and secrete less as soluble fragments than do controls. Surface labeling demonstrated that there is little APP or CD62 on the surface of resting platelets. Upon activation, control platelets exhibited more of both proteins on their surface, while advanced AD patients exhibited similar amounts of CD62 as controls, but retained significantly more surface APP. AD platelets secreted similar amounts of most soluble alpha-granule contents as controls, but less APP fragments. Together these results suggest a processing defect that may account for greater deposition of A beta-containing products in the vasculature to which activated platelets adhere.