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Biomedical subjects

S P Bajaj

Publications and source records attributed to S P Bajaj.

At least 19 recordsLinked to original sources
PubMed2007

A cross-reactive material positive variant of coagulation factor XI (FXIP520L) with a catalytic defect.

Inherited deficiency of the trypsin-like protease factor (F) XI is associated with a mild to moderate bleeding diathesis. In most cases, FXI protein is reduced in plasma, and examples of dysfunctional circulating FXI variants are rare. We characterized the defect in one such variant with a proline to leucine substitution at residue 520. FXI Pro520 corresponds to chymotrypsin Pro161, and is conserved in most members of the chymotrypsin protease family. Recombinant FXI containing this substitution will be referred to as FXI(P161L). k(cat) for cleavage of chromogenic substrates and for activation of the natural FXIa substrate FIX is approximately 3-fold lower for activated FXI(P161L) (FXIa(P161L)) than for wild-type FXIa (FXIa(WT)), consistent with an abnormal protease active site. Inhibition of FXIa(P161L) by diisopropyl fluorophosphate is 2.4-fold slower than for FXIa(WT), suggesting distortion of the protease oxyanion hole. Binding to p-aminobenzamidine, a probe for the integrity of the S1 substrate-binding site, was similar for FXIa(WT) and FXIa(P161L). Rates of carbamylation of Ile16 were also similar for FXIa(WT) and FXIa(P161L), indicating that the critical salt bridge between Ile16 and Asp194 forms normally during protease activation. Cumulatively, the data demonstrate that Pro161 is required for normal active site oxyanion hole conformation in FXIa. Examination of the FXIa crystal structure and modeling studies indicate that Pro161 forms several hydrophobic contacts with adjacent amino acids that stabilize active site conformation. Leucine can be incorporated at position 161 in FXIa, but would not form the extensive stabilizing network of hydrophobic interactions formed by Pro161.

Aspartic Acid↗
PubMed2006

Reconstruction of abdominal wall by whole thigh flap.

Closure of extensive abdominal wall defects can be a very challenging task as there are no known large local or free vascularized flaps available that could cover the entire abdomen. Tensor fascia latae (TFL) has been widely used for abdominal wall reconstruction [Hill HL, Nahai F, Vasocnez LO. The tensor fascia lata myocutaneous free flap. Plast Reconstr Surg 1978;61:517-22]. However, the dimensions of the standard TFL flap limit its use in cases of large full thickness abdominal wall defects. Therefore, we have used an ingenious technique of raising the entire thigh skin as a fasciocutaneous flap (whole thigh flap) based on the concept of fusion of angiosomal territories, to reconstruct such a defect following excision of a large abdominal wall tumour.

Abdominal Wall↗
PubMed2005

The first epidermal growth factor-like domains of factor Xa and factor IXa are important for the activation of the factor VII--tissue factor complex.

During tissue factor (TF)-induced coagulation, the factor (F)VIIa-TF complex activates factor (F)X and factor (F)IX. Through positive feedback, the generated FXa and FIXa activate FVII-TF. The first epidermal growth factor-like (EGF1) domains of FX and FIX serve as important TF-recognition motifs when FVIIa-TF activates FX or FIX. Here, we investigated the role of EGF1 domains of FXa and FIXa during the activation of FVII-TF and inhibition by tissue factor pathway inhibitor (TFPI). FXaPCEGF1 (EGF1 domain of FXa replaced with that of protein C), and FXaQ49P (EGF1 domain mutant with impaired calcium-binding), and the corresponding FIXa mutants were generated, and their abilities to activate FVII-TF were compared with the wild-type (WT) enzymes. In the absence of TF, the rates of FVII activation were similar between WT enzymes and mutant FXa and FIXa proteases. In the presence of either soluble TF (sTF) or relipidated TF, each mutant of FXa or FIXa activated FVII-TF at a slower rate than the corresponding WT enzyme. Kinetics of inhibition of the amidolytic activity of WT and the mutant FXa proteases by either two-domain or full-length TFPI were similar. However, compared with the complex of TFPI-FXaWT, the abilities of the complexes of TFPI-FXa mutants to inhibit FVIIa-TF were impaired. We conclude that the EGF1 domains of FXa and FIXa are important for the activation of FVII-TF and for the formation of FVIIa-TF-FXa-TFPI complex.

Amino Acid Motifs↗
PubMed2001

Factor IXa:factor VIIIa interaction. helix 330-338 of factor ixa interacts with residues 558-565 and spatially adjacent regions of the a2 subunit of factor VIIIa.

The physiologic activator of factor X consists of a complex of factor IXa, factor VIIIa, Ca(2+) and a suitable phospholipid surface. In one study, helix 330 (162 in chymotrypsin) of the protease domain of factor IXa was implicated in binding to factor VIIIa. In another study, residues 558-565 of the A2 subunit of factor VIIIa were implicated in binding to factor IXa. We now provide data, which indicate that the helix 330 of factor IXa interacts with the 558-565 region of the A2 subunit. Thus, the ability of the isolated A2 subunit was severely impaired in potentiating factor X activation by IXa(R333Q) and by a helix replacement mutant (IXa(helixVII) in which helix 330-338 is replaced by that of factor VII) but it was normal for an epidermal growth factor 1 replacement mutant (IXa(PCEGF1) in which epidermal growth factor 1 domain is replaced by that of protein C). Further, affinity of each 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Glu-Gly-Arg-IXa (dEGR-IXa) with the A2 subunit was determined from its ability to inhibit wild-type IXa in the tenase assay and from the changes in dansyl fluorescence emission signal upon its binding to the A2 subunit. Apparent K(d(A2)) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) approximately 100 nm, dEGR-IXa(R333Q) approximately 1.8 micrometer, and dEGR-IXa(helixVII) >10 micrometer. In additional experiments, we measured the affinities of these factor IXa molecules for a peptide comprising residues 558-565 of the A2 subunit. Apparent K(d(peptide)) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) approximately 4 micrometer, and dEGR-IXa(R333Q) approximately 62 micrometer. Thus as compared with the wild-type or PCEGF1 mutant, the affinity of the R333Q mutant for the A2 subunit or the A2 558-565 peptide is similarly reduced. These data support a conclusion that the helix 330 of factor IXa interacts with the A2 558-565 sequence. This information was used to model the interface between the IXa protease domain and the A2 subunit, which is also provided herein.

Amino Acid Substitution↗
PubMed2001

Structure and biology of tissue factor pathway inhibitor.

Human tissue factor pathway inhibitor (TFPI) is a modular protein comprised of three Kunitz type domains flanked by peptide segments that are less structured. The sequential order of the elements are: an N-terminal acidic region followed by the first Kunitz domain (K1), a linker region, a second Kunitz domain (K2), a second linker region, the third Kunitz domain (K3), and the C-terminal basic region. The K1 domain inhibits factor VIIa complexed to tissue factor (TF) while the K2 domain inhibits factor Xa. No direct protease inhibiting functions have been demonstrated for the K3 domain. Importantly, the Xa-TFPI complex is a much more potent inhibitor of the VIIa-TF than TFPI by itself. Furthermore, the C-terminal basic region of TFPI is required for rapid physiologic inhibition of coagulation and is needed for the inhibition of smooth muscle cell proliferation. Although a number of additional targets for attachment have been reported, the C-terminal basic region appears to play an important role in binding of TFPI to cell surfaces. A primary site of TFPI synthesis is endothelium and the endothelium-bound TFPI contributes to the antithrombotic potential of the vascular endothelium. Further, increased levels of plasma TFPI under septic conditions may represent endothelial dysfunction. We have proposed that the extravascular cells that synthesize TF also synthesize TFPI providing dual components necessary for the regulation of clotting in their microenvironment. Like the TF synthesis in these cells is augmented by serum, so is the case with the TFPI gene expression. TFPI gene knock out mice reveal embryonic lethality suggesting a possible role of this protein in early development. Since TF-induced coagulation is thought to play a significant role in many disease states, including disseminated intravascular clotting, sepsis, acute lung injury and cancer, recombinant TFPI may be a beneficial therapeutic agent in these disease states to attenuate pathologic clotting. The purpose of this review is to outline recent developments in the field related to the structural specificity and biology of TFPI.

Acute Disease↗
PubMed2000

Thermodynamic linkage between the S1 site, the Na+ site, and the Ca2+ site in the protease domain of human coagulation factor xa. Studies on catalytic efficiency and inhibitor binding.

The serine protease domain of factor Xa (FXa) contains a sodium as well as a calcium-binding site. Here, we investigated the functional significance of these two cation-binding sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na(+) binds to the substrate bound FXa with K(d) approximately 39 mm in the absence and approximately 9.5 mm in the presence of Ca(2+). Sodium-bound FXa (sodium-Xa) has approximately 18-fold increased catalytic efficiency ( approximately 4.5-fold decrease in K(m) and approximately 4-fold increase in k(cat)) in hydrolyzing S-2222 (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide), and Ca(2+) further increases this k(cat) approximately 1.4-fold. Ca(2+) binds to the protease domain of substrate bound FXa with K(d) approximately 705 microm in the absence and approximately 175 microm in the presence of Na(+). Ca(2+) binding to the protease domain of FXa (Xa-calcium) has no effect on the K(m) but increases the k(cat) approximately 4-fold in hydrolyzing S-2222, and Na(+) further increases this k(cat) approximately 1.4-fold. In agreement with the K(m) data, sodium-Xa has approximately 5-fold increased affinity in its interaction with p-aminobenzamidine (S1 site probe) and approximately 4-fold increased rate in binding to the two-domain tissue factor pathway inhibitor; Ca(2+) (+/-Na(+)) has no effect on these interactions. Antithrombin binds to Xa-calcium with a approximately 4-fold faster rate, to sodium-Xa with a approximately 24-fold faster rate and to sodium-Xa-calcium with a approximately 28-fold faster rate. Thus, Ca(2+) and Na(+) together increase the catalytic efficiency of FXa approximately 28-fold. Na(+) enhances Ca(2+) binding, and Ca(2+) enhances Na(+) binding. Further, Na(+) enhances S1 site occupancy, and S1 site occupancy enhances Na(+) binding. Therefore, Na(+) site is thermodynamically linked to the S1 site as well as to the protease domain Ca(2+) site, whereas Ca(2+) site is only linked to the Na(+) site. The significance of these findings is that during physiologic coagulation, most of the FXa formed will exist as sodium-Xa-calcium, which has maximum biologic activity.

4-Aminobenzoic Acid↗
PubMed2000

Protease nexin-2/Amyloid beta-protein precursor regulates factor VIIa and the factor VIIa-tissue factor complex.

Protease nexin-2/amyloid beta-protein precursor (PN-2/AbetaPP) and its Kunitz protease inhibitory (KPI) domain were characterized as inhibitors of factor VIIa (FVIIa) and factor VIIa-tissue factor complex (FVIIa-TF). PN-2/AbetaPP and KPI domain inhibited FVIIa with an apparent K(i) of 1.1+/-0.2x 10(-7) M and 1.5+/-0.1x10(-7) M, respectively. When soluble tissue factor (TF(1-219)) was present, there was increased FVIIa inhibition by PN-2/AbetaPP or KPI domain (K(i)=7.8+/-0.3x10(-8) M and 6.8+/-0.6x10(-8) M, respectively). When relipidated tissue factor (TF(1-243)) was present, the K(i) of FVIIa inhibition by PN-2/AbetaPP increased 4.7-fold further. PN-2/AbetaPP complexed with FVIIa, as shown on gel filtration and solid phase binding assay. The apparent second-order rate constant of inhibition of FVIIa by PN-2/AbetaPP in the absence of TF(1-219) was less than that of the FVIIa-TF(1-219) complex. Antithrombin in the absence of TF(1-219) also had a lower apparent second-order rate constant of inhibition than in its presence. In a mixture that included FVIIa, relipidated TF(1-243) and factor X, PN-2/AbetaPP or KPI domain had an IC(50) at 65 and 250 nM, respectively; antithrombin and heparin (1 U/mL) had an IC(50) of 12.8 nM. These data indicate that tissue factor promoted the inhibition of FVIIa by PN-2/AbetaPP or KPI domain, but antithrombin was a better inhibitor of soluble FVIIa-TF in extrinsic tenase.

Amyloid beta-Protein Precursor↗
PubMed2000

Abdominoperineal electrical injury involving urogenital organs.

Although the incidence of electrical burns is not very high this incidence is rising in India. The residual effects of electrical burns are sometimes very severe and long lasting, if not managed properly. This case report is of a patient who sustained severe electrical burns over his left arm, lower abdomen and perineal area. The injuries were so severe that life-saving disarticulation of the limb together with excision of the involved pubic bone and genitalia and suprapubic urinary diversion was done. During follow up an unusual finding was noticed. There was a peritubal leak with no drainage from the catheter and on removal of the catheter, a manual examination of the urinary bladder revealed a stone which was removed at the same time. Following removal of the stone two ureteric openings with the trigone were seen and it was concluded that the granulating tissue was actually the posterior wall of the bladder. This was then mobilized and reposited into the abdomen. The deficient lower abdomen was reconstructed with a tensor fascia lata flap. A penile reconstruction for the patient is under consideration.

Abdominal Injuries↗
PubMed2000

Large transverse fasciocutaneous leg flap: whole leg flap.

Large defects of leg and sole often need massive tissue transfer. As an alternative to microvascular transfer, we have developed a fasciocutaneous flap in which almost the whole of the skin of the opposite leg is transferred based just on the septocutaneous perforators of the posterior tibial artery. The flap has been used as a cross-leg fasciocutaneous flap with potential for use as a free flap by taking a segment of the posterior tibial artery. Our experience with 11 cases is presented. Two patients suffered marginal necrosis while donor site problems were seen in another two patients. We have found this flap to be safe, technically easy and with minimal donor-site morbidity.

Adolescent↗
PubMed1999

Protease and EGF1 domains of factor IXa play distinct roles in binding to factor VIIIa. Importance of helix 330 (helix 162 in chymotrypsin) of protease domain of factor IXa in its interaction with factor VIIIa.

Previous studies revealed that cleavage at Arg-318-Ser-319 in the protease domain autolysis loop of factor IXa results in its diminished binding to factor VIIIa. Now, we have investigated the importance of adjacent surface-exposed helix 330-338 (162-170 in chymotrypsin numbering) of IXa in its interaction with VIIIa. IXWT, eight point mutants mostly based on hemophilia B patients, and a replacement mutant (IXhelixVII in which helix 330-338 is replaced by that of factor VII) were expressed, purified, and characterized. Each mutant was activated normally by VIIa-tissue factor-Ca2+ or XIa-Ca2+. However, in both the presence and absence of phospholipid, interaction of each activated mutant with VIIIa was impaired. The role of IXa EGF1 domain in binding to VIIIa was also examined. Two mutants (IXQ50P and IXPCEGF1, in which EGF1 domain is replaced by that of protein C) were used. Strikingly, interactions of the activated EGF1 mutants with VIIIa were impaired only in the presence of phospholipid. We conclude that helix 330 in IXa provides a critical binding site for VIIIa and that the EGF1 domain in this context primarily serves to correctly position the protease domain above the phospholipid surface for optimal interaction with VIIIa.

Binding Sites↗
PubMed1999

New insights into how blood clots: implications for the use of APTT and PT as coagulation screening tests and in monitoring of anticoagulant therapy.

Blood coagulation occurs efficiently on cell surfaces such as activated platelets and monocytes, and fibroblasts. It is initiated by limited amounts of tissue factor (TF) exposed at the sites of vascular injury that complexes with trace amounts of circulating factor VIIa (FVIIa). Additional FVIIa-TF complexes are formed from FVII-TF involving positive feedback loops, including FVIIa-TF as well as factors Xa and IXa as they are formed in subsequent steps. For sustained normal coagulation to proceed, effective in vivo activation of factor X requires the participation of factor IXa generated via the FVIIa-TF complex. This may, in part, be due to effective inhibition of factor Xa and FVIIa-TF complex by tissue factor pathway inhibitor that results in blockage of direct activation of factor X by the FVIIa-TF complex. Additional generation of factor Xa at injury sites may then proceed via the FIXa-VIIIa pathway. Thrombin generated from prothrombin via complex formation of prothrombin with FXa and FVa on phospholipid surfaces (prothrombinase complex) powerfully accelerates coagulation by activation of FVIII and FV, and sustains coagulation through activation of FXI. Thus, in light of our current understanding of how blood clots in vivo, it is clear that both prothrombin time (PT) and activated partial thromboplastin time (APTT) are highly artificial in vitro systems with major limitations. Nevertheless, these tests are quite useful as global screening tests for abnormalities in the intrinsic or extrinsic, as well as common, pathways of coagulation and for monitoring of anticoagulant therapy.

Anticoagulants↗
PubMed1999

Transcriptional expression of tissue factor pathway inhibitor, thrombomodulin and von Willebrand factor in normal human tissues.

Under normal physiologic conditions, tissue factor pathway inhibitor (TFPI) is synthesized primarily by the microvascular endothelium. Using Northern blotting, we studied its transcriptional expression in different organs and compared it with the expression of two other endothelial specific proteins, namely thrombomodulin (TM) and von Willebrand factor (vWF). The order of mRNA expression for each protein was: TFPI-placenta>lung>liver>kidney>heart>skeletal muscle> or =pancreas>brain; TM-heart>pancreas>lung>skeletal muscle>kidney> or =liver>placenta>brain; and vWF-heart>skeletal muscle>pancreas>lung> or =kidney>placenta>brain>liver. Notably, heart expressed TM and vWF mRNA in large amounts and only small amounts of TFPI whereas lung expressed all three mRNAs in significant amounts. Placenta, on the contrary, expressed large amounts of TFPI but only small amounts of TM and vWF mRNAs. Brain by this technique was found to express undetectable amounts of TFPI and TM mRNAs but small amounts of vWF mRNA. The expression of TFPI mRNA in the brain was however detected by RT/PCR and the antigen was localized to the endothelium of microvessels as well as to the astrocytes and oligodendrocytes. Since ultimate expression of proteins is linked to the expression of their mRNAs, our data support a concept that vascular endothelium is made up of phenotypically diverse groups of cells and that endothelial cells of different vascular beds express specific sets of genes that enable them to carry out tissue-specific functions. Importantly, since astrocytes are known to express tissue factor, the TFPI expression by these cells may control coagulation in their microenvironment and their response to injury and inflammation.

Brain↗
PubMed1999

Region of factor IXa protease domain that interacts with factor VIIIa: analysis of select hemophilia B mutants.

Essential to hemostasis is the interaction of factor IXa with factor VIIIa. Recent studies indicate that helix-330 in the protease domain of factor IXa provides a critical binding site for factor VIIIa. Although weaker interactions cannot be ruled out, a primary role of the EGF1 domain of factor IXa in this context may be to serve as a spacer in properly positioning the factor IXa protease domain for optimal interaction with factor VIIIa. The role of the Gla domain, as well as of the EGF2 domain of factor IXa, in binding to factor VIIIa is not clear. The region of factor VIIIa that interacts with the protease domain of factor IXa is quite possibly located in the A2 domain. Furthermore, it should be noted (Table 1) that the corresponding helix residues in factor VIIa bind to tissue factor, and, in factor Xa, they are involved in binding to factor Va. Thus, a common function of this helix (162 in chymotrypsin numbering) in several blood coagulation proteases may be to serve as an anchoring point for the respective cofactor.

Animals↗