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Biomedical subjects

V V Kakkar

Publications and source records attributed to V V Kakkar.

At least 19 recordsLinked to original sources
PubMed1995

The integrin alpha IIb beta 3 contains distinct and interacting binding sites for snake-venom RGD (Arg-Gly-Asp) proteins. Evidence that the receptor-binding characteristics of snake-venom RGD proteins are related to the amino acid environment flanking the sequence RGD.

We have previously demonstrated [Lu, Williams, Deadman, Salmon, Kakkar, Wilkinson, Baruch, Authi and Rahman (1994) Biochem. J. 304, 929-936] the preferential antagonism of the interactions of the integrin alpha IIb beta 3 on activated platelets with three immobilized glycoprotein ligands (fibrinogen, fibronectin and von Willebrand factor) by a selected panel of snake-venom RGD (Arg-Gly-Asp)-containing proteins including the disintegrins kistrin and elegantin, and the neurotoxin variant dendroaspin. Kistrin and dendroaspin, although structurally unrelated, contain similar amino acids flanking the tripeptide RGD and behaved as identical antagonists preferentially inhibiting platelet adhesion to immobilized fibrinogen as opposed to fibronectin. In contrast, elegantin, which shares extensive sequence similarity with kistrin but has different amino acids around the tripeptide RGD, preferentially inhibited platelet adhesion to immobilized fibronectin as opposed to fibrinogen. To develop further insights into the mechanisms underlying the preferential antagonism shown by the venom proteins in the adhesion studies, we, in the present study, sought to determine the binding properties of kistrin, elegantin and dendroaspin to the alpha IIb beta 3 complex by radioligand kinetic and competition studies. In direct binding experiments, both kistrin and dendroaspin were observed to bind to a single class of binding site on ADP-activated platelets with apparent equilibrium dissociation constant (Kdapp) values of 42 +/- 2 nM and 21 +/- 6 nM respectively. In competition studies, dendroaspin blocked the binding of 125I-labelled kistrin to ADP-activated platelets in a simple competitive manner, with an apparent equilibrium inhibition constant (Kiapp) of 143 +/- 14 nM, from which an indirect Kdapp = 22 nM for dendroaspin was determined. This result suggests that kistrin and dendroaspin bind to the same site on the integrin alpha IIb beta 3 consistent with their similar inhibitory properties. In contrast, elegantin recognized two classes of binding sites on the alpha IIb beta 3 complex with Kdapp values of 10.5 +/- 0.8 nM and 175 +/- 10 nM, and, unlike dendroaspin, did not inhibit the binding of 125I-labelled kistrin to ADP-activated platelets. However, in reciprocal experiments both kistrin and dendroaspin inhibited the binding of 125I-elegantin to ADP-activated platelets in a non-competitive manner, with Kiapp values of 34 +/- 3 nM and 21 +/- 2 nM respectively. Thus elegantin appears to interact with distinct but interacting sites on the alpha IIb beta 3 complex from the binding site of kistrin and dendroaspin, consistent with its distinctive inhibitory preferences as shown in platelet adhesion studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1995

Detection and characterization of seven novel protein S (PROS) gene lesions: evaluation of reverse transcript-polymerase chain reaction as a mutation screening strategy.

The molecular genetic analysis of protein S deficiency has been hampered by the complexity of the protein S (PROS) gene and by the existence of a homologous pseudogene. In an attempt to overcome these problems, a reverse transcript-polymerase chain reaction (RT-PCR) mutation screening procedure was developed. However, the application of this mRNA-based strategy to the detection of gene lesions causing heterozygous type I protein S deficiency appears limited owing to the high proportion of patients exhibiting absence of mRNA derived from the mutation-bearing allele ("allelic exclusion"). Nevertheless, this strategy remains extremely effective for rapid mutation detection in type II/III protein S deficiency. Using the RT-PCR technique, a G-to-A transition was detected at position +1 of the exon IV donor splice site, which was associated with type I deficiency and resulted in both exon skipping and cryptic splice site utilization. No abnormal protein S was detected in plasma from this patient. A missense mutation (Asn 217 to Ser), which may interfere with calcium binding, was also detected in exon VIII in a patient with type III protein S deficiency. A further three PROS gene lesions were detected in three patients with type I deficiency by direct sequencing of exon-containing genomic PCR fragments: a single base-pair (bp) deletion in exon XIV, a 2-bp deletion in exon VIII, and a G0to-A transition at position -1 of the exon X donor splice site all resulted in the absence of mRNA expressed from the disease allele. Thus, the RT-PCR methodology proved effective for further analysis of the resulting protein S-deficient phenotypes. A missense mutation (Met570 to Thr) in exon XIV of a further type III-deficient proband was subsequently detected in this patient's cDNA. No PROS gene abnormalities were found in the remaining four subjects, three of whom exhibited allelic exclusion. However, the father of one such patient exhibiting allelic exclusion was subsequently shown to carry a nonsense mutation (Gly448 to Term) within exon XII.

Base Sequence↗
PubMed1995

Long-term chemotaxis studies on adherent cells: effect of platelet-derived growth factor-BB on human vascular smooth muscle cell migration.

Several chemotaxis methods have been developed which allow the study of different aspects of cell migration. The major limitation of such methods is the lack of a sustained chemotactic signal. Long-term chemotaxis phenomena which are known to take place in vivo have remained largely uninvestigated. Ways to maintain sustained chemotactic signals were sought and the used to investigate the long-term chemotactic effect of platelet-derived growth factor BB (PDGF-BB) on human vascular smooth muscle cells (HVSMC). PDGF-BB was adsorbed onto microcarrier beads and then embedded in agar. PDGF-BB diffusion was slow and a high and sustained local concentration was maintained in the agar. When PDGF-BB-loaded beads embedded in agar were placed at the edge of a tissue culture dish with HVSMC plated in the center, preferential movement was observed in the direction of the PDGF-BB source. This method was subsequently used to study directional movement of HVSMC arising from explants. This report demonstrates that PDGF-BB if present in an anisotropic concentration induces directional cell movement from such explants. By allowing the study of the effect of sustained chemotactic signals upon cultured cells or cells arising from explants, this method may provide a suitable model for investigating in vivo chemotaxis phenomena.

Cell Movement↗
PubMed1995

Polymorphic variation in the human protein C (PROC) gene promoter can influence transcriptional efficiency in vitro.

The haplotype frequencies of two closely linked diallelic polymorphisms in the protein C(PROC) gene promoter were determined in the British population. In principle, differences in transcription efficiency between PROC promoter haplotypes could represent an additional risk factor in determining whether or not an individual already predisposed to venous thrombosis will come to clinical attention. In order to explore this postulate, transient transfection of human hepatoma cells with PROC promoter-luciferase reporter gene constructs was performed in vitro. In HepG2 cells, the T.....A haplotype exhibited at least a two-fold higher transcription efficiency than the C.....G haplotype. A sample of British protein C deficiency patients with recurrent venous thrombosis was then allelotyped by a combination of oligonucleotide discriminant hybridization and DNA sequencing. The frequency of the low expressing C.....G haplotype was found to be slightly yet not significantly higher in these patients (0.43) than in controls (0.35).

Base Sequence↗
PubMed1995

Population differences in the frequency of the factor V Leiden variant among people with clinically symptomatic protein C deficiency.

The factor V Leiden variant, responsible for the phenomenon of activated protein C resistance, was found to be less frequent among British (0.06) and Swedish/Danish (0.15) protein C deficiency patients than previously reported in a Dutch study (0.19). In the Swedish population, a significantly increased frequency of the factor V Leiden allele was apparent in protein C deficiency patients as compared to healthy controls. However, this was not found in the British population. Coinheritance of the factor V Leiden variant is therefore unlikely to be the sole determinant of whether a person with protein C deficiency will come to clinical attention. Nevertheless, when patient data were analysed by type of protein C deficiency, it was noted that the frequency of the factor V Leiden variant was 2.8-fold higher in type II patients compared to type I patients. A possible explanation of this disparity is discussed.

Alleles↗
PubMed1994

Preferential antagonism of the interactions of the integrin alpha IIb beta 3 with immobilized glycoprotein ligands by snake-venom RGD (Arg-Gly-Asp) proteins. Evidence supporting a functional role for the amino acid residues flanking the tripeptide RGD in determining the inhibitory properties of snake-venom RGD proteins.

The inhibitory properties of a panel of snake-venom-derived RGD (Arg-Gly-Asp) proteins, including the disintegrins kistrin, elegantin and albolabrin, and the neurotoxin homologue dendroaspin, were investigated in a platelet-adhesion assay using three immobilized ligands of the glycoprotein IIb-IIIa complex (alpha IIb beta 3), namely fibrinogen, fibronectin and von Willebrand factor (vWF). The snake-venom proteins preferentially inhibited the adhesion of ADP-treated platelets to one or more of the immobilized ligands. Kistrin and dendroaspin exhibited similar inhibitory characteristics, abrogating platelet adhesion to fibrinogen and vWF at nanomolar concentrations, but poorly inhibiting adhesion to fibronectin. Kistrin and dendroaspin share little overall amino-acid-sequence identity, but a considerable level of sequence similarity exists around the RGD tripeptide. Synthetic cyclic peptides corresponding to these regions of kistrin and dendroaspin inhibited platelet adhesion to both fibrinogen and fibronectin with approximately equal potency, but were 100-fold weaker antagonists of the interactions of the alpha IIb beta 3 complex with fibrinogen than their parent proteins. The disintegrins elegantin and albolabrin, which share approx. 60% overall amino-acid-sequence similarity with kistrin but have different residues around the RGD tripeptide, exhibited different antagonistic preferences. Elegantin inhibited platelet adhesion to immobilized vWF and fibronectin, but was significantly less effective at disrupting adhesion to fibrinogen. Albolabrin selectively inhibited platelet adhesion to immobilized vWF and was less effective with fibrinogen and fibronectin as adhesive ligands. In contrast with the behaviour of these venom proteins, the adhesion of ADP-treated platelets to immobilized fibrinogen, fibronectin and vWF was inhibited non-selectively by a range of monoclonal antibodies with specificity for the alpha IIb beta 3 complex. These observations, therefore, define antagonistic preferences in this panel of venom proteins towards the interactions of the alpha IIb beta 3 complex with three immobilized glycoprotein ligands.

Amino Acid Sequence↗
PubMed1992

Thrombin-induced proliferation and expression of platelet-derived growth factor-A chain gene in human vascular smooth muscle cells.

Treatment of human vascular smooth muscle cells (SMC) with human alpha-thrombin greatly increased DNA synthesis and cell proliferation. Both the integrity of the catalytic site and that of the anion binding exosite were required for expression of this activity. Experiments employing Northerns indicated induction of c-fos expression as well as a time-dependent induction of platelet-derived growth factor-A (PDGF-A) gene by thrombin. The thrombin mitogenic activity was potentiated by PDGF-BB, insulin and the vasoconstrictor peptide endothelin-1 suggesting synergism by convergence of intracellular growth-promoting signals. SMC treatment with pertussis toxin and forskolin indicated that the mitogenic activity of thrombin may be induced via signal transduction mechanism(s) involving changes in cAMP levels and activation of a Gi-like protein. These results suggest that thrombin may play a functional role in the regulation of human vascular SMC proliferation.

Aorta↗
PubMed1992

Use of low molecular weight heparin in pregnancy.

In a controlled study of 15 pregnant patients undergoing therapeutic termination of pregnancy, seven received subcutaneously 5,000 anti-FXa units of low molecular weight (LMW) heparin 15 and 3 h prior to the termination, and eight patients acted as controls. Paired maternal and fetal blood samples were taken (before or immediately after the termination) for assay of heparin activity by a chromogenic anti-FXa method sensitive to levels of 0.02 anti-FXa U/ml. LMW heparin was detected in all maternal samples of the test patients but was not detected in any of the fetal samples. The use of LMW heparin as a thromboprophylactic agent was then evaluated in 11 patients who were known to have a severe thromboembolic tendency, had suffered recurrent miscarriages and had responded poorly to conventional anticoagulation (oral anticoagulant, conventional heparin). All patients receiving LMW heparin in thromboprophylactic doses completed uneventful pregnancies and gave birth to healthy babies (three for the first time) without complication. Bone density scans performed in all patients shortly after the delivery showed normal mineral mass. We conclude that LMW heparin does not cross the placental barrier, and in addition offers satisfactory antithrombotic protection for both maternal and placental circulation. In addition, this study provides preliminary data from 11 patients suggesting LMWH may not give rise to maternal osteoporosis, a finding that now needs further investigation.

Abortion, Habitual↗
PubMed1992

Synthesis and biological activity of ketomethylene pseudopeptide analogues as thrombin inhibitors.

Ketomethylene pseudopeptide analogues Aa-Pro-Arg psi (COCH2) Gly-pip, 1, where Aa are D- or L-amino acids (Dpa, beta, beta-diphenylalanine; alpha Nal, alpha-naphthylalanine; beta Nal, beta-naphthylalanine; Fgl, fluorenylglycine) with highly lipophilic side chains and psi (COCH2) is a ketomethylene pseudopeptide bond, have been synthesized through a modified Dakin-West reaction under very mild conditions with a high yield using tripeptide 4 with a labile functional group directly on the side chain. Their enzymatic assay of thrombin inhibition has been carried out. The structure-activity relationship study indicated that a lipophilic side chain on the amino acid in the P3 position is very important for binding to the apolar site of thrombin. Compound 1a with D-Dpa at the P3 position has a Ki of 0.2 microM and it doubles thrombin clotting time at only 3 times higher concentration. These values are about 7 times better than those of the corresponding D-Phe analogues. Furthermore, 1a shows poor inhibitory activity against plasmin, factor Xa, urokinase, and kallikrein. Preliminary in vivo testing (3-4-kg rabbit as the animal model) shows no observable side effect (change of blood pressure and accumulation of blood platelet in lungs) at a dose of 1 mg/kg.

Peptides↗
PubMed1992

Non-specific influence of chemical modification upon the properties of antithrombin III:modification of carboxyl groups.

The ability of antithrombin III to inhibit thrombin was observed to be rapidly inactivated upon specific modification of carboxyl groups. The loss of activity, upon treatment with nitrotyrosyl ester in the presence of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate, was concomitant to the incorporation of 2 moles of nitrotyrosine per mole of inhibitor indicating the modification of only two carboxyl groups. Moreover, the modification occurred with loss, also, of the ability of the native protein to bind tightly to heparin. The modified antithrombin III retained a reduced affinity for heparin (eluting at 0.3M NaCl from heparin Agarose) and was observed to be a competitive inhibitor of the heparin-dependent rate of inhibition of thrombin by native antithrombin III. However, FAB-MS (fast atom bombardment mass spectroscopy) analysis of digests of modified material gave no indication that modification was localized to specific Asp or Glu residues. It is concluded that the loss of activity is due to deleterious change in conformation during modification. These findings, together with our previous report upon tryptophan modification of antithrombin III [1] suggest that the nature of the molecule is such that considerable care must be taken in interpretation of results when investigating the structure/function relationships of this protein by chemical modification.

Antithrombin III↗
PubMed1992

Generation of forms of fragment E with differing thrombin-binding properties during digestion of fibrinogen by plasmin.

Two principal forms of fragment E are generated upon digestion of fibrinogen by plasmin, according to the concentration of enzyme used. At a high concentration of plasmin (above 10 micrograms/ml), a form lacking fibrinopeptide A (FpA) at the N-terminus of the A alpha-chain was generated. This form of fragment E caused a dose-dependent increase in thrombin clotting times but had no measurable inhibitory activity towards thrombin cleavage of D-phenylalanyl-L-pipecolyl-L-arginine p-nitroanilide. At a low concentration of plasmin (less than 1 microgram/ml), fragment E containing 35-40% of the original amount of FpA was present in the terminal digest. The FpA-containing form of fragment E inhibited thrombin cleavage of fibrinogen, inhibited amidolytic activity and bound to the enzyme with an affinity 3-fold tighter than fibrinogen itself (Kd 4.1 +/- 0.3 microM as opposed to 12.7 +/- 1.8 microM). During digestion of fibrinogen at low plasmin concentration, up to 65% of the FpA was cleaved just subsequent to the progressive release of B beta-(1-42)-peptide, and the Arg-16-Gly-17 bond of the A alpha-chain became relatively stable towards plasmin action when present in fragment E (and possibly fragment Y). It is proposed that both forms of fragment E can inhibit clotting by binding to the fibrin(ogen)-recognition site (anion-binding exosite) of thrombin. The FpA-containing form of fragment E can also inhibit binding that occurs distal to the P1 site and thereby interfere with amidolysis of the peptide substrate. Our finding of a lability of the Arg-16-Gly-17 bond in the early phase of digestion may provide an alternative explanation of the increased FpA concentrations observed during thrombolytic therapy.

Binding Sites↗
PubMed1992

Mastoparan promotes exocytosis and increases intracellular cyclic AMP in human platelets. Evidence for the existence of a Ge-like mechanism of secretion.

Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, accelerates guanine nucleotide exchange and GTPase activity of purified GTP-binding proteins. In the present study we have examined the functional consequences of exposure of intact human platelets to mastoparan. Mastoparan promoted rapid (less than or equal to 1 min) dose-dependent increases in 5-hydroxy[14C]tryptamine and beta-thromboglobulin release from dense-granule and alpha-granule populations respectively. The exocytotic response did not result from a lytic effect of mastoparan and occurred in the complete absence of platelet shape change and aggregation. Liberation of [3H]arachidonate and increases in cytosolic [Ca2+] (detected with fura 2) were not observed in platelets stimulated with mastoparan. Similarly, in platelets preloaded with [3H]inositol during reversible electroporation, mastoparan did not cause the accumulation of [3H]inositol phosphates. Mastoparan-induced secretion was unaffected by preincubation with either the protein kinase C inhibitor staurosporine (10 nM-10 microM) or prostacyclin (PGI2; 100 ng/ml) and was not accompanied by phosphorylation of the 45 kDa protein kinase C substrate or the 20 kDa protein normally associated with platelet activation. The G-protein inhibitor guanosine 5'-[beta-thio]diphosphate (GDP[S]; 1 mM) attenuated the secretion induced by mastoparan in both intact and saponin-permeabilized platelets. Encapsulation of GDP[S] during reversible permeabilization inhibited mastoparan-induced secretion, providing evidence for an intracellular action of GDP[S]. In all these studies thrombin (0.05-0.2 unit/ml) elicited characteristic responses, and thrombin-induced secretion was inhibited by staurosporine, PGI2 and GDP[S]. Mastoparan also increased intra-platelet cyclic AMP in a dose-dependent manner. Mastoparan and PGI2 increased 32P incorporation into a protein of approx. 24 kDa, whereas phosphorylation of a 50 kDa substrate was only seen in PGI2-stimulated platelets. These results indicate that mastoparan promotes secretion by a mechanism which does not involve stimulation of phospholipase C and suggest that the secretory event may result either from a direct fusogenic action of mastoparan and/or from stimulation of the putative exocytosis-linked G-protein, Ge.

Alkaloids↗
PubMed1992

Prenatal exclusion of haemophilia A and carrier testing by direct detection of a disease lesion.

A novel mutation was detected in the Factor VIII gene of a sporadic case of severe haemophilia A. The lesion, a CGA-->TGA transition, converts Arg 795 to Term and adequately accounts for the severe phenotype observed. PCR/direct sequencing was used to confirm the carrier status in the mother. Exclusion of haemophilia A in an at-risk pregnancy was then achieved by demonstration of the absence of this lesion in fetal DNA from a chorionic villus sample. The mutation was also detectable by chemical cleavage of mismatch (CCM), which both confirmed the prenatal diagnosis and established the carrier status of the proband's sister. This example therefore serves to illustrate the potential of direct gene analysis in sporadic cases of haemophilia A and/or in families uninformative for known RFLPs.

Base Sequence↗
PubMed1992

Protein C deficiency and thromboembolism: recurrent mutation at Arg 306 in the protein C gene.

A CGA----TGA transition in the protein C gene, resulting in an Arg306----Term substitution, was detected in a Swedish kindred with thrombotic disease whose members exhibit plasma protein C activity/antigen levels consistent with type I protein C deficiency. Although an identical lesion has been reported previously in several Dutch families, RFLP typing indicated that the Dutch and Swedish mutations were unlikely to be identical by descent and probably arose by recurrent mutation.

Amino Acid Sequence↗
PubMed1992

Omission of exon 12 in cystic fibrosis transmembrane conductance regulator (CFTR) gene transcripts.

Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA transcripts isolated from both expressing and "non-expressing" cell types of normal individuals exhibit differential splicing to a variable extent in a region encoding the putative nucleotide binding fold of the CFTR polypeptide. Sequence analysis of the aberrant fragments obtained after cDNA polymerase chain reaction amplification confirmed the in-frame joining of exons 11 and 13. The proportion of alternative splicing is reproducible and constant in a given individual. The omission of exon 12 in a significant proportion of transcripts supports the hypothesis that a minimal amount of correctly expressed CFTR is sufficient for the maintenance of a clinically normal phenotype.

Base Sequence↗
PubMed1992

A novel homozygous missense mutation in the protein C (PROC) gene causing recurrent venous thrombosis.

A novel homozygous CCC----CTC (Pro 247----Leu) substitution was detected in the protein C genes of a patient, born to consanguineous parents, with inherited type 1 protein C deficiency and recurrent venous thrombosis. Since one of four heterozygous relatives was also clinically affected, the condition appears to be inherited as an incompletely recessive trait in this family.

Base Sequence↗