Role of protein kinases of human red cell membrane in deformability and aggregation changes

The proteomic analysis has showed that red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation changes. Exposure of red blood cells (RBCs) to some chemical compounds led to change in the RBC microrheological properties. When forskolin (10 microM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p < 0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 26%; p < 0.01). Red cell aggregation (RBCA) was significantly decreased under these conditions (p < 0.01). Markedly less changes of deformability was found after RBC incubation with protein kinase stimulator C (PKC)--phorbol 12-myristate 13-acetate (PMA). This drug reduced red cell aggregation only slightly. It was inhibited red cell tyrosine phosphotase activity by N-vanadat and was obtained a significant RBCD rise and RBCA lowering. The similar effect was found when cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor--lavendustin eliminated the above mention effects. On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways.     

The role of red blood cell adenylyl cyclase activation in changes of erythrocyte membrane microrheological properties

The ability of red blood cells (RBCs) for the reversible change of their shape under passing through capillaries in microcirculation mainly depends on membrane elasticity of these cells. Phosphorylation of some membrane proteins can result in the changes of microrheological red blood cell properties. Here we show a significant increase in RBC deformability (RBCD) after incubation with isoproterenol (10⁻⁶ M). Red blood cell aggregation (RBCA) decreased under these conditions only slightly. When forskolin (10 μM), an adenylyl cyclase (AC) stimulator, was added to the RBC suspension, RBCD increased significantly (p < 0.05). Some more changes of deformability were found after incubation of RBC with stable penetrating analog of cyclic adenosine phosphate (cAMP), dibutyryl-cAMP, (dB-cAMP, 50 μM) and after phosphodiesterase (PDE) activity inhibition with Vinpocetine, Rolipram, or IBMX. It was found that Gs-proteins inhibitor Clonidine and specific Gi-protein stimulator Mastaparan 7 increased both RBCD and RBCA. On the whole, the data clearly show that the RBC aggregation and deformation changes are related with activation of the different intracellular signaling pathways. We suppose that RBCD increase was mainly associated with activation of the adenylyl-cyclase-cAMP system.     

Alteration of red blood cell microrheology by anti-tumor chemotherapy drugs

The aim of this study was to estimate effects of some chemotherapy drugs on the elasticity and deformability of the membrane of a red blood cell (RBC). It was found that incubation of red blood cells (RBCs) with cisplatin or epoetin alpha led to considerable (by 10–17%; p < 0.05) increase in the RBC deformability and that cisplatin could activate tyrosine protein kinases (TPKs). Preincubation of RBCs with a specific inhibitor of EGF-R and Src kinase, lavendustin A, almost completely prevented the cisplatin effect. Tyrosine phosphatase inhibitor, sodium orthovanadate, increased the RBC deformability (p < 0.05). This effect was also abandoned by lavendustin A. To test a hypothesis on the involvement of protein kinases of mature RBCs in control of their membrane elasticity, the cells were incubated with phorbol 12-myristate 13-acetate (PMA) activating protein kinase Cα (PKCα). PMA increased the RBC deformability only moderately (by 8%, p < 0.05) and the effect was canceled by nonselective and selective PKC inhibitors staurosporin and 4-(1-methylindol-3-yl)maleimide hydrochloride. Erythropoietin is known to inhibit the nonselective cation channels of the RBC membrane; however, preincubation of the cells with verapamil did not cancel the increase in their deformability. Hence, this increase in deformability could be a result of the action of tyrosine protein kinases, the more so that this effect was almost completely canceled by lavendustion A. The results suggest that the presence of functionally active protein kinases and phosphatases in the membranes of mature RBC makes them a target for the addressed effects of signal molecules, including some chemotherapy drugs, causing consecutive alterations in the RBC membrane elasticity, microrheological properties, and transport potential.     

Aggregation of erythrocytes and their membranes flexibility in patients with cancer-associated anemia: Mechanisms of changes under the influence of epoetin alfa

The relationships between the red blood cell (RBC) membrane elasticity and RBC aggregation in healthy individuals and in patients with anemia of malignant tumors treated with human erythropoietin drug epoetin alfa (EA) were analyzed. It was found that prior to the treatment of patients, incubation of RBCs with EA was accompanied by an increase of RBC deformability and the reduction of their aggregation (RBCA). In these circumstances the two characteristics of the RBC microrheology correlated negatively with each other (r =–0.734, p < 0.05). In contrast, aggregation and deformability of RBCs from healthy individuals increased under the influence of EA and positively correlated with each other (r = 0.580, p < 0.05). After a 4-week treatment of patients with EA, aggregation response of the patients’ RBCs was increased by 29% (p < 0.05) and was close to that of healthy RBCs. This change of the RBC aggregation response may be connected with an alteration of the sensitivity of the membrane cationic channel to EA and an increase of the cell deformability. This possibility was supported by experiments with the use of Ca²⁺-channel blocker verapamil and Ca²⁺-chelating agent EDTA. Under these conditions a decrease of the RBC aggregation varied from 40 to 50% (p < 0.05). It was suggested that the effectors of calcium regulatory cascade upon exposure to EA may be membrane integrin receptors of type IIb–IIIa. This assumption was confirmed by experiments employing the inhibitors of these receptors (tirofibam and integrelin) and a preparation of monoclonal antibodies against IIb–IIIa receptors (monafram), which produced a significant decrease (20–30%, p < 0.05) of the RBC aggregation. Thus, our findings suggest that the altered aggregation response of RBCs in anemic patients with malignant tumors can be restored by the correction of anemia with epoetin alfa.     

酪氨酸蛋白激酶和蛋白激酶C对N-乙酰氨基葡萄糖转移酶V的双重调控

在人肝癌细胞７７２１中研究了酪氨酸蛋白激酶（ＴＰＫ）和蛋白激酶Ｃ（ＰＫＣ）的激活剂［分别为表皮生长因子（ＥＧＦ）和佛波酯（ＰＭＡ）］和各种蛋白激酶抑制剂对Ｎ－乙酰氨基葡萄糖转移酶Ｖ（ＧｎＴ－Ｖ）活力的影响,以探讨ＴＰＫ和ＰＫＣ对ＧｎＴ－Ｖ的调节。结果发现,ＥＧＦ或ＰＭＡ处理细胞４８ｈ后,ＧｎＴ－Ｖ的活力明显增高;蛋白激酶的非特异性抑制剂槲皮素和染料木黄酮（ｇｅｎｉｓｔｅｉｎ）在抑制ＴＰＫ和ＰＫＣ的同时,抑制ＧｎＴ－Ｖ的基础活力,并完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的增高作用;ＴＰＫ的特异性抑制剂Ｔｙｒｐｈｏｓｔｉｎ－２５和ＰＫＣ的特异性抑制剂Ｄ－鞘氨醇分别应用时,各自只能部分地取消ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导。但当Ｔｙｒｐｈｏｓｔｉｎ－２５和Ｄ－鞘氨醇同时加入培养基中则可完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导激活。蛋白质合成抑制剂环己亚胺和蛋白激酶抑制剂作用相仿,不但可抑制ＧｎＴ－Ｖ的基础活力,也可完全消除ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的激活。以上结果提示ＥＧＦ或ＰＭＡ通过蛋白激酶调节ＧｎＴ－Ｖ的酶蛋白合成,并且ＧｎＴ－Ｖ受到膜性ＴＰＫ和ＰＫＣ的双重调节,其中ｍ－ＴＰＫ较ｍ－ＰＫＣ更为重要。     

Treadmill exercise suppresses muscle cell apoptosis by increasing nerve growth factor levels and stimulating p-phosphatidylinositol 3-kinase activation in the soleus of diabetic rats

We investigated the effects of treadmill exercise performed regularly for 6 weeks on the levels of nerve growth factor (NGF), tyrosine kinase A and p75 receptors, phosphatidylinositol 3-kinase (PI3-K), mitogen-activated protein kinase/extracellular signal-regulated kinase (Erk) 1,2, cyclic AMP response element-binding protein (CREB), and caspase-3 in the soleus of rats with streptozotocin (STZ)-induced diabetes. Thirty-two male Sprague–Dawley rats were divided into the following four groups: (1) normal control group (NCG; n = 8), (2) normal exercise group (NEG; n = 8), (3) diabetes control group (DCG; n = 8), and (4) diabetes exercise group (DEG; n = 8). Diabetes was induced by intraperitoneal injection of STZ (55 mg/kg dissolved in 0.05 M citrate buffer, pH 4.5). Rats were subjected to treadmill exercise 5 days a week for 6 weeks. The protein level of NGF significantly increased in the NEG and DEG (p < 0.001), whereas the levels of tyrosine kinase A and p75 receptors significantly increased in the NEG (p < 0.001). The levels of t-PI3-K, p-PI3-K, and p-CREB, and the p-CREB/t-CREB ratio significantly increased in the NEG (p < 0.001, respectively). The p-PI3-K/t-PI3-K ratio significantly increased in the DEG (p < 0.001). The p-Erk1/t-Erk1 ratio significantly increased in the NEG (p < 0.001), whereas the p-Erk2/t-Erk2 ratio significantly decreased in the DCG and DEG (p < 0.001). The caspase-3 level significantly increased in the DCG compared with that in the DEG (p < 0.001). These results suggest that treadmill exercise increases NGF levels and accelerates p-PI3-K activation in order to suppress apoptotic cell death in the soleus muscle of diabetic rats.     

The effect of exercise and zinc supplement on the hematological parameters in rats

This study evaluates the consequences of a session of intensive short-duration exercise and Zn supplementation on different hematological variables. Forty male Wistar rats were divided into four groups (n=10): the first nonsupplemented, maintained at rest (R); the second nonsupplemented, undergoing exercise (E); the third supplemented with Zn, kept at rest (ZnR); and the fourth supplemented with Zn, undergoing exercise (ZnE). Zinc supplements (200 ppm) were given in drinking water. The exercise consisted of a single session of swimming until exhaustion. At rest, RBC, Hb, and Hto fell (p<0.05), whereas red cell indices, MCV, and MCH rose (p<0.05) in +ZnR compared with R; MCHC remained unchanged (ZnR vs R). After exercise, RBC, Hb, and Hto increased significantly in E and in ZnE compared with R and ZnR, respectively. In addition, RBC and Hb were lower (p<0.01) in ZnE compared with E; however, MCV and MCH were higher (p<0.05) in the group ZnE vs E. With respect to white blood cells—leukocytes (WBC), limphocytes (LYMPH), and neutrophiles (NEUT)—no significant differences were observed between groups at rest (ZnR vs R). WBC and LYMPH increased significantly in E with respect to the rest situation (E vs R), but this did not happen in supplemented animals (ZnE vs ZnR). Level of pH decreased after exercise both in E and in ZnE, but the fall was lower in the latter. We believe that a single session of swimming until exhaustion leads to an increase in RBC, Hb, and Hto without causing changes in MCV, MCH, and MCHC. On the other hand, Zn supplementation leads to an increase of MCV and MCH, although they remain within normal levels. Furthermore, this supplementation produces lower metabolic acidoses after exercise that leads to leukocyte stability.     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.     

Cellular determinants of low-shear blood viscosity

Low-shear viscometry is one of the methods commonly used to estimate the degree of red blood cell (RBC) aggregation in various bloods and RBC suspensions. However, it has been previously shown that alterations in RBC morphology and mechanical behavior can affect the low-shear apparent viscosity of RBC suspensions; RBC aggregation is also sensitive to these cellular factors. This study used heat treatment (48°C, 5 min), glutaraldehyde (0.005–0.02%) and hydrogen peroxide (1 mM) to modify cell geometry and deformability. Red blood cell aggregation was assessed via a Myrenne Aggregometer (“M” and “Ml” indexes), RBC suspension viscosity was measured using a Contraves LS-30 viscometer, and RBC shape response to fluid shear stresses (i.e., deformability) was determined by ektacytometry (LORCA system). Our results indicate that low-shear apparent viscosity and related indexes may not always reflect changes of RBC aggregation if cellular properties are altered: for situations where RBC aggregation has been only moderately affected, cellular mechanical factors may be the major determinant of low-shear viscosity. These findings thus imply that in situations which may be associated alterations of RBC geometry and/or deformability, low-shear viscometry should not be the sole measurement technique used to assess RBC aggregation.     

In vitro influence of zinc and magnesium on the deformability of red blood cells artificially hardened by heating

Trace elements have been shown to improve red blood cell (RBC) deformability: zinc in sickle cell disease and magnesium in an in vitro model of chemically rigidified erythrocytes. In this study, we investigated the effect and the influence of incubation time of zinc or magnesium on an in vitro model of rigidified RBCs by heating. Erythrocyte rigidity was determined by viscosimetry at high shear rate by a falling ball viscosimeter MT 90. In the first part of the study, six normal volunteers participated. Viscosimetry was performed on native blood before and after heating the sample for 10 min at 50°C. Therefore, increasing concentrations of zinc gluconate (final concentration: 0.5–4 g/L) or isotonic NaCl as control medium were added to the sample. Heating induced a twofold increase in all indices of RBC rigidity (p<0.05). At all these concentrations of zinc, a highly significant, dose-related fluidifying effect was observed (40–70%): this effect was immediately obtained and did not change over 60 min. Even at the highest concentration, recovery was not complete. In the second part of the study, we studied magnesium’s effects on blood. In a first protocol, whole blood was rigidified by heating at 56°C for 10 min, and the correcting effect of 5 min of incubation at 37°C of RBCs in 150 mmol/L NaCl, MgSO₄, magnesium acetate, and magnesium gluconate was investigated. In a second protocol, the same incubation with NaCl and magnesium salts was made on blood that had not been previously heated. In a third protocol, the correcting effect of magnesium gluconate on heated red blood cells was tested at four concentrations (75, 150, 225, and 300 mmol/L) over 1 h, for evaluating the effects of both concentration and time. Erythrocyte rigidity by heating is corrected by the three salts employed in protocol 1 (compared to sodium). In protocol 2, the deformability of normal (nonheated) red cells is not modified by magnesium. In protocol 3, no marked modification over 1 h is observed. The correcting effect is not complete for 75 mmol/L Mg, but remains the same at the three other concentrations. This study shows that zinc and magnesium at supraphysiological concentration are able to reverse RBC’s rigidification induced by heating, but that magnesium does not modify the flexibility of normal RBCs. This article suggests that zinc and magnesium may be studied in vivo as potential pharmacologic tools for improving hemorheologic disturbances.     

Identification, purification and reconstitution of thiamin metabolizing enzymes in human red blood cells.

Thiamin and its mono- (TMP), di- (TDP) and triphosphate (TTP) were assayed in adult human whole blood using high-performance liquid chromatography (HPLC). TDP and TTP were detected in red blood cells (RBC), but not in plasma. After incubation with 20 microM thiamin and 5 mM glucose for 2 h, the TDP and TTP contents of RBC increased from 111 to 222 and 0.6 to 2.2 nmol/l of packed RBC, respectively, suggesting enzymatic conversion of thiamin to TDP and then to TTP. Thiamin pyrophosphokinase (TPK, EC 2.7.6.2) had not been isolated before from human materials, nor had cytosolic adenylate kinase (AK1, EC 2.7.4.3) in human RBC been demonstrated to catalyze the phosphorylation of TDP to TTP, although AK1 from pig and chicken skeletal muscle possess TTP-synthesizing activity. TPK and AK1 in a human RBC lysate were therefore purified by a series of the conventional techniques. The specific activity of the purified TPK, which was obtained as a single protein, was 720 nmol TDP formed/mg protein per h at 37 degrees C. A partially purified AK1 preparation catalyzed the formation of TTP from TDP (specific activity, 170 nmol/mg protein per h at 37 degrees C) in addition to its proper reaction to form ATP from ADP. After incubation of the purified TPK and AK1 with 20 microM thiamin in the presence of ATP, ADP and Mg2+ at 37 degrees C for 48 h, the amounts of TDP and TTP synthesized were 465 and 54.0 pmol/250 microliters reaction mixture, respectively. Neither TDP nor TTP was formed when TPK was omitted from the reaction mixture and an omission of AK1 resulted in the formation of TDP alone. These results indicate that thiamin is converted to TDP by TPK and, subsequently, to TTP by AK1 in human RBC.     

Detection of tyrosine-specific protein kinases with gastrin as exogenous substrate

Gastrin was recently shown to be phosphorylated on its single tyrosine by the epidermal growth factor (EGF)-stimulated tyrosine protein kinase (TPK). The TPK previously detected in the murine lymphoma (LSTRA) induced by the Moloney murine leukemia virus phosphorylates gastrin, the apparent K_m is 65 μM and the maximum rate 1900 pmol/min per mg; the kinase is more efficeint with MnCl₂ than with MgCl₂, is stimulated by NaVO₃ and inhibited by ZnCl₂. Gastrin phosphorylation is observed only when a TPK is expressed by the cell: extracts of fibroblasts infected with a temperature-sensitive mutant of the Rous sarcoma virus had no gastrin kinase activity when grown at the non-permissive temperature whereas cells grown at the permissive temperature were transformed and disclosed a clear gastrin kinase activity. Gastrin kinases were detected in various transformed cells; human lymphomas, K562 cells, cells from a patient with acute proliferative leukemia, and normal cels; human T and B lymphocytes.     

Effect of carbonyl compounds on red blood cells deformability

The effect of Maillard reaction on red blood cells (RBC) deformability was investigated. Exposure of RBC to carbonyl compounds (dl-glyceraldehyde, glyoxal, glycolaldehyde, 3-deoxyglucosone, and d-glucose) leading to Maillard reaction caused a marked decrease in RBC deformability even at 1 mM level. The decrease rate depended on the kind of carbonyl compounds, in which both dl-glyceraldehyde and glyoxal significantly decreased the RBC deformability (p < 0.05). In addition, the decrease rate also differed among volunteers tested, indicating that the sensitivity against carbonyl compounds varies among them. In order to elucidate the mechanism of the decrease in RBC deformability, RBC was exposed to carbonyl compounds in the presence of aminoguanidine which is the inhibitor of AGE formation in Maillard reactions. Aminoguanidine inhibited the decrease in RBC deformability by dl-glyceraldehyde and glyoxal. When Hb which has a high reactivity with carbonyl compounds was incubated with those carbonyl compounds, dl-glyceraldehyde and glyoxal showed the high reactivity with Hb compared with other carbonyl compounds. These results indicate that Maillard reaction between RBC proteins and carbonyl compounds leads to the decrease in RBC deformability. On the other hand, generated by carbonyl compounds involved in lowering the deformability only to a negligible level.     

Biochemical and Clinical Improvement of Cytotoxic State by Amantadine Sulphate

  1. The main idea of the open clinical trial was to compare the income and outcome clinical picture and the evolution of the biochemical markers in the defined intervals in closed head injury group patients.2. In the group of 32 patients, mean age 40.78±15.36 years suffering from closed traumatic brain injury the following markers were measured: glycaemia, malondialdehyde (MDA) as marker of lipid peroxidation, beta-caroten, total SH groups as marker of protein oxidation in the following intervals: between the 1st and the 3rd, between the 3rd and the 7th, between the 1st and the 7th day respectively.3. Glycaemia significantly decreased since the 1st day till the 3rd day (p < 0.05) and since the 1st day till the 7th day (p < 0.05) but it was not significantly changed since the 3rd day till the 7th day (p > 0.05).4. MDA 1st × MDA 3rd p > 0.05 insignificant change, MDA 1st × MDA 7th p < 0.001—high significant decrease, MDA 3rd × MDA 7th—p < 0.0001—very high significant decrease.5. Beta-caroten the 1st × beta-caroten the 3rd day was insignificantly changed—p > 0.05, the 3rd × the 7th day beta-caroten increased significantly—p < 0.0002, the 1st day × 7th day beta-caroten significantly increased—p < 0.0001.6. We examined the SH groups only in nine patients, due to technical problems and SH groups decrease on the 3rd day (p < 0.005).7. In 18 amantadine sulphate subgroups (randomly selected), there was 5.5% lethality and mean outcome GCS (outGCS) 9.83±3.8, while lethality of the control subgroup (n=14) was 42.9%, mean outGCS 6.28±3.5.     

Harpin induces mitogen-activated protein kinase activity during defence responses in Arabidopsis thaliana suspension cultures

Elicitation of Arabidopsis thaliana (L.) Heynh. suspension cultures with the bacterial protein harpin (from Pseudomonas syringae pv. syringae) induced the activation of two kinases of 39 and 44 kDa, as demonstrated by in-gel kinase assays using myelin basic protein (MBP) as a substrate. Both these kinases appeared to be tyrosine-phosphorylated upon activation, as demonstrated by treatment with tyrosine phosphatase and immunoprecipitation using an anti-phosphotyrosine monoclonal antibody. An inhibitor of mammalian mitogen-activated protein kinase (MAPK) activation, PD98059, inhibited harpin-induced MBPK activation, but did not inhibit the activity of these kinases. PD98059 also inhibited harpin-induced programmed cell death and defence gene expression, suggesting the involvement of harpin-induced MAPKs in defence responses in Arabidopsis thaliana. Received: 23 February 1999 / Accepted: 22 July 1999     

佛波酯引起蛋白激酶C下降调节的专一性

探讨了佛波酯（ＰＭＡ）对蛋白激酶的下降调节是否有激酶专一性及亚型专一性．用组蛋白Ｈ１作为蛋白激酶Ｃ（ＰＫＣ）和蛋白激酶Ａ（ＰＫＡ）的受体底物,加入ＰＫＣ和ＰＫＡ的特异性激活剂区分ＰＫＣ和ＰＫＡ,用聚谷酪（４１）为酪氨酸蛋白激酶（ＴＰＫ）的专一性受体底物,以３２Ｐ－ＡＴＰ为３２Ｐ共同供体底物测定三种蛋白激酶的活力,并用免疫组化法测定ＰＫＣ亚型．结果发现ＰＭＡ对人７７２１肝癌细胞只引起ＰＫＣ而不引起ＰＫＡ和ＴＰＫ的下降调节,ＰＫＣ的非特异性抑制剂槲皮素和特异性抑制剂Ｄ－鞘氨醇能大部分取消ＰＭＡ对ＰＫＣ的下降调节,但ＴＰＫ抑制剂ｇｅｎｅｓｔｅｉｎ则没有阻断下降调节的作用．用ＨＬ－６０细胞还证明ＰＭＡ只对含量丰富的ＰＫＣα和ＰＫＣβⅡ亚型而不对含量很少的ＰＫＣβⅠ亚型发生下降调节．上述结果说明ＰＭＡ对蛋白激酶的下降调节有激酶和亚型专一性．     

Interactions of the human red cell membrane tyrosine kinase with heparin

Heparin interacts with protein kinases in various ways; the different patterns of behavior of heparin towards protein kinases contributes to the characterization of these enzymes. We studied the interactions between heparin and a new type of tyrosine kinase extracted from the normal human red cell membrane. We found that heparin inhibited kinase activity by competition with ATP. Furthermore the interaction of heparin with the red cell membrane tyrosine kinase allowed us to use heparin-agarose chromatography as a step towards tyrosine kinase purification.     

Role of hemoglobin oxygenation in the modulation of red blood cell mechanical properties by nitric oxide

It has been previously demonstrated that both externally generated and internally synthesized nitric oxide (NO) can affect red blood cell (RBC) deformability. Further studies have shown that the RBC has active NO synthesizing mechanisms and that these mechanisms may play role in maintaining normal RBC mechanical properties. However, hemoglobin within the RBC is known to be a potent scavenger of NO; oxy-hemoglobin scavenges NO faster than deoxy-hemoglobin via the dioxygenation reaction to nitrate. The present study aimed at investigating the role of hemoglobin oxygenation in the modulation of RBC rheologic behavior by NO. Human blood was obtained from healthy volunteers, anticoagulated with sodium heparin (15 IU/mL), and the hematocrit was adjusted to 0.4 L/L by adding or removing autologous plasma. Several two mL aliquots of blood were equilibrated at room temperature (22 ± 2 °C) with moisturized air or 100% nitrogen by a membrane gas exchanger, The NO donor sodium nitroprusside (SNP), at a concentration range of 10^?7–10^?4 M, was added to the equilibrated aliquots which were maintained under the same conditions for an additional 60 min. The effect of the non-specific NOS inhibitor l-NAME was also tested at a concentration of 10^?3 M. RBC deformability was measured using an ektacytometer with an environment corresponding to that used for the prior incubation (i.e., oxygenated or deoxygenated). Our results indicate an improvement of RBC deformability with the NO donor SNP that was much more pronounced in the deoxygenated aliquots. SNP also had a more pronounced effect on RBC aggregation for deoxygenated RBC. Conversely, l-NAME had no effect on deoxygenated blood but resulted in impaired deformability, with no change in aggregation for oxygenated blood. These findings can be explained by a differential behavior of hemoglobin under oxygenated and deoxygenated conditions; the influence of oxygen partial pressure on NOS activity may also play a role. It is therefore critical to consider the oxygenation state of intracellular hemoglobin while studying the role of NO as a regulator of RBC mechanical properties.