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V Kakkar

Publications and source records attributed to V Kakkar.

18 recordsLinked to original sources
PubMed2001

Arg-Tyr-Asp (RYD) and Arg-Cys-Asp (RCD) motifs in dendroaspin promote selective inhibition of beta1 and beta3 integrins.

Arg-Gly-Asp (RGD) is a unique minimal integrin-binding sequence that is found within several glycoprotein ligands. This sequence has also been found in snake-venom anti-platelet proteins, including the disintegrins and dendroaspin, a natural variant of short-chain neurotoxins isolated from the venom of Dendroaspis jamesonii. In the present study, the motifs RYD and RCD were introduced into the dendroaspin scaffold to replace RGD. Both motifs in dendroaspin caused inhibition of ADP-induced platelet aggregation with IC(50) values of 200 and 300 nM respectively, similar to that of the wild-type RGD motif (170 nM). In comparison with wild-type dendroaspin, both RYD- and RCD-containing dendroaspins were more selective in the inhibition of the adhesion of K562 cells to laminin rather than to fibrinogen and fibronectin, even though they were 10-30-fold less potent at inhibiting K562 cell (containing alpha(5)beta(1) integrin) adhesion to laminin compared with wild-type. Interestingly, the RYD motif produced a similar IC(50) value to the RGD motif at inhibiting A375-SM cell (beta(3) integrin) adhesion to collagen, whereas the RCD motif was approx. 2-6-fold less potent compared with either RGD or RYD. These findings show that the selectivity of dendroaspin binding to beta(1) and beta(3) integrins can be modulated by the introduction of alternative cell recognition sequences.

Amino Acid Motifs↗
PubMed2001

Evaluation of the role of proline residues flanking the RGD motif of dendroaspin, an inhibitior of platelet aggregation and cell adhesion.

The effect of a panel of proline mutants of dendroaspin, an inhibitor of platelet aggregation and cell adhesion, including A(42)-dendroaspin, A(47)-dendroaspin, A(49)-dendroaspin, A(42,47)-dendroaspin and A(47,49)-dendroaspin, was investigated using platelet-aggregation and cell-adhesion assays. Here we show that a single alanine-for-proline substitution did not affect potency when measured as the ability either to inhibit platelet aggregation induced by ADP (IC(50) approximately 170 nM) or to block transfected A375-SM cell adhesion to fibrinogen in the presence of Mn(2+) as compared with wild-type dendroaspin. By comparison, double proline substitution with alanines significantly reduced the potency in both assays by approx. 5-8-fold. These observations, therefore, suggest that proline residues flanking the RGD motif in dendroaspin and other RGD-containing venom proteins, e.g. disintegrins, may contribute to maintaining a favourable conformation for the solvent-exposed RGD site for its recognition by integrin receptors.

Amino Acid Motifs↗
PubMed2000

Molecular genetic analysis of severe protein C deficiency.

Severe protein C deficiency is a rare, early onset, venous thrombotic condition that is inherited as an autosomal recessive trait. The protein C (PROC) genes of nine unrelated individuals with severe protein C deficiency were sequenced yielding a total of 13 different lesions. Eight of these were novel, including a gross gene deletion, three missense mutations, two micro-deletions, a splicing mutation and a single base-pair substitution in the HNF-3 binding site in the PROC gene promoter. Evidence for the pathogenicity of the mutations detected was obtained by molecular modelling, in vitro splicing assay and reporter gene assay. Neither the plasma protein C activity level nor the nature of the PROC gene lesions detected were found to be a good prognostic indicator of the age of onset or clinical severity of thrombotic symptoms. Other factors may thus complicate the relationship between genotype and clinical phenotype. Indeed, in two patients, the inheritance of either one or two Factor V Leiden alleles in addition to two PROC gene lesions could have served to precipitate the thrombotic events. No association was however apparent between clinical severity and the possession of a particular promoter polymorphism genotype. Despite the absence of a clear genotype-phenotype relationship, the molecular genetic analysis of the severe recessive form of protein C deficiency potentiates both the counselling of affected families and the provision of antenatal exclusion diagnosis.

Alternative Splicing↗
PubMed1997

Coagulation factor abnormalities after the Fontan procedure and its modifications.

OBJECTIVE: Recently we reported the prevalence of thromboembolism in patients who underwent the Fontan procedure and its modifications. Although hemodynamic factors may well contribute to thromboembolism, recent evidence suggests that coagulation factor abnormalities may also play a role. We therefore set out to investigate the coagulation status in a group of patients who had undergone the Fontan procedure. METHODS: The study population consists of 20 children who had undergone the Fontan procedure and its modifications. They were examined for coagulation factor abnormalities. Concentrations of serum albumin, total protein, and liver enzymes were also measured. The median age at the time of the operation was 6.2 years (17 months to 8 years) with a male/female ratio of 2.3:1. The median time from the Fontan repair was 4.9 years (18 to 76 months). RESULTS: Protein C (p < 0.001), protein S (p < 0.02), and factor VII (p < 0.001) were significantly lower than the normal range. The changes in serum albumin and total protein and factors II, IX, and X were not significant. CONCLUSIONS: It is possible that deficiency in protein C, protein S, and factor VII partly account for the prevalence of thromboembolism after Fontan-type repairs. The risk of long-term anticoagulation should be weighed against the best palliative procedure for these patients. We suggest that reduced protein C, protein S, and factor VII levels in this group of patients should be regarded as risk factors and that such patients should be treated with anticoagulants.

Child↗
PubMed1997

The routine determination of the endogenous thrombin potential, first results in different forms of hyper- and hypocoagulability.

The area under the thrombin generation curve (the endogenous thrombin potential; ETP) has been proposed as a parameter for plasma-based hypercoagulability and to monitor anticoagulant treatment. We present an ETP assay for the routine laboratory using a centrifugal analyser. Throughput is 30 samples/h, within and between run imprecision is 4-5.6%. Suitable substrates were developed for the ranges of 10-500% and 2-100% of normal. Independent of tissue factor concentration (if > 4 pM), the normal value of the extrinsic ETP is 384.8 +/- 51.7 nM.min. The intrinsic ETP, triggered by ellagic acid, is 414 +/- 41 nM.min. The ETP is decreased to 15 and 35% of normal by oral anticoagulation (INR 2.5-4.0) and by heparin administration (APTT 1.5-2.5 x control). The ETP is increased in untreated subjects with congenital antithrombin deficiency and in women using oral contraceptives. In deep vein thrombosis (phlebographically confirmed), it is increased by 29.4% (extrinsic) and 53% (intrinsic). In (angiographically assessed) coronary artery disease the increase is by 10% and 17% respectively.

Adolescent↗
PubMed1993

Nasal mucociliary clearance & mucus pH in patients with diabetes mellitus.

Nasal mucociliary clearance (NMC) time and nasal mucus pH were studied in 50 patients suffering from diabetes mellitus and in a group of 50 healthy non-smokers and non-alcoholic controls. NMC time and pH values were found to be significantly increased in diabetics (NMC = 18.02 +/- 5.08 and pH = 7.96 +/- 0.75) as compared to controls (NMC = 7.49 +/- 1.06 and pH 6.43 +/- 0.67). The increase in NMC and pH was much more in patients having insulin dependent diabetes mellitus (IDDM) (NMC - 20.87 +/- 4.71 and pH -8.38 +/- 0.56) than in non-insulin dependent diabetes mellitus (NIDDM) (NMC - 15.16 +/- 3.67 and pH -7.53 +/- 0.687) and also when the duration of disease was more than 10 yr (NMC - 22.36 +/- 4.36 and pH -8.47 +/- 0.607). This impairment was attributed to osmotic diuresis with loss of water and electrolytes from all parts of the body and also small vessel abnormalities encountered in diabetes.

Adolescent↗
PubMed1992

Long term effects of tamoxifen. Biological effects of Tamoxifen Working Party.

A total of 153 breast cancer patients who participated in two trials of adjuvant tamoxifen and who had not recurred were recruited into a study of the long term effects of tamoxifen. There were 60 controls (no tamoxifen), 73 ex-users (mostly for 2 years) and 20 current users (median treatment duration 72 months) and the median follow-up time was 7 years. A wide ranging study of lipids, hormones, bone density and haemostasis was undertaken. When compared with controls, current users had lower cholesterol levels (especially low density cholesterol), and increased triglyceride levels. Thyroid hormones were higher and sex hormone binding globulin was almost doubled. Bone density was non-significantly higher, clotting times were slightly shorter and fibrinogen and antithrombin III levels were reduced. However few of these changes persisted in ex-users, suggesting that most of the biological effects of treatment are reversible on cessation of treatment. This is reassuring for potentially negative side-effects, but also indicates that potentially positive 'side-effects' such as cholesterol lowering only occur while on active treatment.

Bone Density↗
PubMed1990

Prevention of venous thrombosis and pulmonary embolism.

Deep vein thrombosis (DVT) leads to hospitalization for up to 600,000 persons each year in the United States. Venous thrombosis in itself may be benign, but the condition can lead to dangerous complications and has a high recurrence rate. Strategies to prevent DVT involve prevention of stasis and reversal of changes in blood coagulability that allow thrombi to form. Pharmacologic agents have been effective in reducing the incidence of DVT and pulmonary embolism. Low-dose subcutaneous heparin is considered a nearly ideal DVT preventative for surgically treated patients. The risk of hemorrhage is the main limitation to routine use of subcutaneous anticoagulants for DVT, but careful patient selection can minimize that risk. After anticoagulant therapy with heparin, generally for 7 to 10 days, oral warfarin is the drug of choice for maintenance anticoagulation to prevent DVT recurrence. Therapy for pulmonary embolism is the same as for DVT--immediate anticoagulation with heparin followed by maintenance with warfarin.

Anticoagulants↗
PubMed1989

Effect of a heparan sulphate with high affinity for antithrombin III upon inactivation of thrombin and coagulation factor Xa.

The kinetics of inhibition of human alpha-thrombin and coagulation Factor Xa by antithrombin III were examined under pseudo-first-order reaction conditions as a function of the concentration of heparan sulphate with high affinity for antithrombin III. The maximum observed second-order rate constant was, for the antithrombin III-thrombin reaction, 1.2 x 10(9) M-1.min-1 compared with 2.4 x 10(9) M-1.min-1 in the presence of high-affinity heparin. However, the maximum rate was catalysed by much higher concentrations of heparan sulphate (1.3 microM) than of heparin (0.025 microM). Differences were also observed in the maximal acceleration of the antithrombin III-Factor Xa interaction: 1.2 x 10(9) M-1.min-1 at 0.2 microM-heparin sulphate compared with 2.2 x 10(9) M-1.min-1 at 0.04 microM-heparin. The differences in properties of heparan sulphate and heparin were analysed by using the random bi-reactant model of heparin action [Griffith (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5460-5464]. It was observed that the apparent binding affinity for thrombin was higher for heparan sulphate (180 nM) than for heparin (14 nM). The rate constant for transformation of the antithrombin III-Factor Xa complex into irreversible product differed between heparan sulphate (96 min-1) and heparin (429 min-1). These properties of the high-affinity heparan sulphate may be of importance in consideration of a putative role in the control of intravascular haemostasis.

Antithrombin III↗
PubMed1983

The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors.

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.

Calcium↗
PubMed1982

Inhibition of human factor Xa by various plasma protease inhibitors.

The inhibitory effects of the plasma protease inhibitors antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin on the activity of human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide susbtrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithromin III, alpha 2-macroglobulin and alpha 1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin were 5.8 . 10(4), 4.00 . 10(4) and 1.36 . 10(4) M -1 . min -1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as alpha 1-antitrypsin greater than antithrombin III greater than alpha 2-macroglobulin in the ratio 4.64: 2.08: 1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.

Antithrombin III↗
PubMedDate not supplied

Effect of adeno-tonsillectomy on hearing threshold and middle ear pressure.

Out of 50 children (100 ears) undergoing adeno-tonsillectomy, 34 ears had hearing threshold 20-50 dB (20 dB is normal) and 32 ears showed negative middle ear pressure of 100 to 400 mmH2O (100 mmH2O is normal). Post-operatively only 7 ears had hearing threshold of 20-30 dB and negative middle ear pressure of 100 to 200 mmH2O. Thus adenoidectomy improves eustachian tube functions.

Acoustic Impedance Tests↗
PubMedDate not supplied

Crystallization test for the detection of head and neck cancer.

Twenty-five patients suffering from head and neck cancer and 25 healthy individuals were subjected to a blood crystallization test with 20% cupric chloride solution and the patterns were compared. In the healthy control group, the typical pattern was an eccentrically placed center of gravity with needles arranged in a radiating fashion, while in the malignant group, there was a transverse bar or transverse formation. The sensitivity of this test procedure was 88%. We therefore conclude that this study is an interesting reliable, economical and above all useful adjunctive for a mass screening programme in the diagnosis of malignant diseases of the head and neck region. The test is based purely on physical phenomena and hence should be performed under controlled physical conditions.

Adult↗
PubMedDate not supplied

Heparin inhibition of human vascular smooth muscle cell hyperplasia.

BACKGROUND: Vascular smooth muscle cell (VSMC) growth is responsible for intimal hyperplasia, a major cause of failure after vascular surgery and angioplasty. Heparin is the first described inhibitor of VSMC growth, but has not proved effective in the prevention of human intimal hyperplasia. Heparin is a heterogeneous substance, which may contain a mixture of components which differ in antiproliferative activity. Isolation of an active component may favourably influence its therapeutic profile. METHODS AND RESULTS: Growth of human VSMC cultured from operative specimens, assessed by cell counting and labelled thymidine incorporation, was used as a model of VSMC proliferation in intimal hyperplasia. Unfractionated (UFH) and low molecular weight (LMWH) heparins inhibit cell growth and thymidine uptake by human VSMCs in response to 15% foetal calf serum. UFHs are more active than LMWHs and this difference increases with increasing heparin dose. To confirm this effect, size-based fractions of heparin were prepared by gel permeation chromatography, and characterised by high performance liquid chromatography. High molecular weight fractions (MW > 21000) have higher activity than fractions of medium (MW 12000-21000) or low molecular weight (MW < 12000). These differences become more pronounced at higher dose, and are statistically significant at 100 micrograms/ml (Mann-Whitney, p < 0.05). CONCLUSIONS: The antiproliferative activity of heparin appears to be maximal in its high molecular weight component.

Cell Division↗