Influence of a calcium dependent protease inhibitor on platelet activation and secretion

Rao, Gundu H. R.; White, James G.; Cox, Chadwick A.

doi:10.1016/0049-3848(87)90101-0

Cited by 14 publications

(3 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Conversely, CI and CII inhibited calpain but not thrombin (Table 1). Consistent with previous studies (Rao et al, 1987;Alonso et al, 1989), leupeptin inhibited the activity of both calpain and thrombin. It is therefore unlikely that the effect of calpain inhibitors on neurite enhancement was mediated by thrombin inhibition.…”

Section: Specificity Of Protease Inhibitorssupporting

confidence: 92%

Multiple Proteases Regulate Neurite Outgrowth in NB2a/dl Neuroblastoma Cells

Shea

Beermann

Nixon

1991

Journal of Neurochemistry

View full text Add to dashboard Cite

Mouse NB2a/dl neuroblastoma cells elaborate axonal neurites in response to various chemical treatments including dibutyryl cyclic AMP and serum deprivation. Hirudin, a specific inhibitor of thrombin, initiated neurite outgrowth in NB2a/dl cells cultured in the presence of serum; however, these neurites typically retracted within 24 h. The cysteine protease inhibitors leupeptin and N-acetyl-leucyl-leucyl-norleucinal (CI; preferential inhibitor of micromolar calpain but also inhibits millimolar calpain) at 10(-6) M considerably enhanced neurite outgrowth induced by serum deprivation, but could not induce neuritogenesis in the presence of serum. A third cysteine protease inhibitor, N-acetyl-leucyl-leucyl-methional (CII; preferential inhibitor of millimolar calpain but also inhibits micromolar calpain), had no detectable effects by itself. Cells treated simultaneously with hirudin and either leupeptin, CI, or CII elaborated stable neurites in the presence of serum. Cell-free enzyme assays demonstrated that hirudin inhibited thrombin but not calpain, CI and CII inhibited calpain but not thrombin, and leupeptin inhibited both proteases. These results imply that distinct proteolytic events, possibly involving more than one protease, regulate the initiation and subsequent elongation and stabilization of axonal neurites. Since the addition of exogenous thrombin or calpain to serum-free medium did not modify neurite outgrowth, the proteolytic events affected by these inhibitors may be intracellular or involve proteases distinct from thrombin or calpain.

show abstract

Section: Specificity Of Protease Inhibitorssupporting

confidence: 92%

Multiple Proteases Regulate Neurite Outgrowth in NB2a/dl Neuroblastoma Cells

Shea

Beermann

Nixon

1991

Journal of Neurochemistry

View full text Add to dashboard Cite

show abstract

“…EDTA displays a high binding constant for zinc and a weak one for calcium and magnesium (about 1.5 times and 2 times, respectively) [11]. So, at the low concen- Leupeptin is also known to have inhibitory activity toward proteases involved in blood coagulation and fibrinolysis [14,15]. In the present study, leupeptin of 100 μ M could prevent blood coagulation, but the protease inhibitor containing 1 μ M leupeptin could not prevent coagulation.…”

Section: Discussionmentioning

confidence: 71%

The Comparison of Parathyroid Hormone Degradation Effect by Various Protease Inhibitors in Blood Specimen

2009

View full text Add to dashboard Cite

Parathyroid hormone (PTH) is released from the parathyroid glands and regulates bone and mineral metabolism. It is a single-chain 84-amino acid polypeptide with a molecular weight of 9,425 Daltons. It is synthesized and secreted by the parathyroid glands, and rapidly cleared from plasma (half-life <5 min). PTH is metabolized to C-terminal fragments by the liver and kidneys, and cleared by the kidney, either as intact PTH or as C-terminal fragments [1]. Background : The objective of this study was to evaluate the role of proteases on the degradation of parathyroid hormone (PTH) in blood samples.Methods : Protease inhibitors with specificity against serine proteases (aprotinin), cysteine proteases (E-64), serine and cysteine proteases (leupeptin), metalloproteases (EDTA), or a protease inhibitor cocktail with a broad spectrum of inhibitory activity were added to blood samples. After storage at room temperature (0-48 hr), PTH levels were measured.Results : PTH levels in samples with the protease inhibitor cocktail did not change significantly after 48 hr of storage at room temperature, but the average PTH levels decreased by 40.7% and 20.1%, in samples stored at room temperature and stored at 4°C without protease inhibitors, respectively. PTH levels in samples with leupeptin were stable for up to 24 hr. After 48 hr, the mean PTH levels decreased by 17.1%, 16.0%, 26.2%, and 32.1%, with 500 KIU/mL aprotinin, 100 μ mol/L leupeptin, 10 μ mol/L E-64, and 10 μ mol/L EDTA, respectively, in the samples stored at room temperature.Conclusions : The decrease in PTH levels in blood samples seemed to be due to the degradation of PTH by proteases. Various proteases, including especially serine proteases, would act together to degrade PTH in blood specimen. The PTH degradation may be inhibited in blood specimen with protease inhibitor cocktail. (Korean J Lab Med 2009;29:104-9)

show abstract

“…In preparation for the present studies, we noted that when leupeptin was added directly into the platelet prothrombinase assay, it slowed the rate of conversion of prothrombin to thrombin. This inhibitory effect on prothrombin conversion was detected even at micromolar concentrations of leupeptin, and likely reflects the known inhibitory action of leupeptin on thrombin (Ruda & Scrutton, 1987;Rao et al, 1987;Brass & Shattil, 1988), decreasing feedback activation by nascent thrombin generated in the prothrombinase assay. Therefore, in order to avoid any unintended effects of leupeptin in the prothrombinase assays in the present study, leupeptin-treated and control platelets were first activated by incubation with the C5b-9 proteins (15 min, 37 °C), and then each was adjusted to the same final leupeptin concentration (20 ^M) upon dilution for assay of prothrombinase activity (see Experimental Procedures).…”

Section: Discussionmentioning

confidence: 80%

Role of calcium and calpain in complement-induced vesiculation of the platelet plasma membrane and in the exposure of the platelet factor Va receptor

Wiedmer¹,

Shattil²,

Cunningham³

et al. 1990

Biochemistry

162

View full text Add to dashboard Cite

The role of calcium and intracellular calpains in the expression of platelet prothrombinase activity was investigated. Incubation of gel-filtered platelets with complement proteins C5b-9 resulted in alpha-granule and dense granule secretion and exposure of membrane binding sites for coagulation factors Va and Xa. This was accompanied by the release of microparticles from the cell surface that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa and the alpha-granule membrane protein GMP-140. Generation of these membrane microparticles was dependent on the presence of extracellular calcium and was accompanied by proteolytic degradation of the cytoskeletal proteins, actin binding protein (ABP), talin, and myosin heavy chain. Microparticle formation was also detected when unstirred platelets were activated by thrombin plus collagen, although proteolysis of ABP, talin, or myosin was not observed. Preincorporation of the calpain inhibitor leupeptin into the platelet cytosol completely blocked C5b-9-induced proteolysis of ABP, talin, and myosin. However, inhibition of this calpain-mediated proteolysis had no effect on platelet secretion, the generation of microparticles, the exposure of membrane sites for factors Va and Xa, or the expression of prothrombinase activity. Furthermore, the microparticles that formed in the presence of leupeptin contained intact ABP, talin, and myosin heavy chain. Prior depletion of ATP with metabolic inhibitors eliminated all platelet responses to thrombin plus collagen, but did not affect C5b-9-induced microparticle formation or exposure of binding sites for factor Va on the microparticles. These data indicate that the formation of microparticles and the expression of platelet prothrombinase activity in response to C5b-9 are dependent upon an influx of calcium into the platelet cytosol, but do not require metabolic energy or calpain-mediated proteolysis of cytoskeletal proteins.

show abstract

Influence of a calcium dependent protease inhibitor on platelet activation and secretion

Cited by 14 publications

References 24 publications

Multiple Proteases Regulate Neurite Outgrowth in NB2a/dl Neuroblastoma Cells

Multiple Proteases Regulate Neurite Outgrowth in NB2a/dl Neuroblastoma Cells

The Comparison of Parathyroid Hormone Degradation Effect by Various Protease Inhibitors in Blood Specimen

Role of calcium and calpain in complement-induced vesiculation of the platelet plasma membrane and in the exposure of the platelet factor Va receptor

Contact Info

Product

Resources

About