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M Jandrot-Perrus

Publications and source records attributed to M Jandrot-Perrus.

At least 19 recordsLinked to original sources
PubMed1997

Adhesion and activation of human platelets induced by convulxin involve glycoprotein VI and integrin alpha2beta1.

We analyzed the interaction of convulxin (Cvx), a 72-kDa protein isolated from the venom of Crotalus durissus terrificus, with human platelets. Cvx is a potent platelet agonist that induces an increase in the intracellular Ca2+ concentration ([Ca2+]i), granule exocytosis and aggregation. 125I-Labeled Cvx binds specifically and rapidly to platelets at binding sites of high and moderate affinity. Platelets adhere to immobilized Cvx in a time-dependent but cation-independent manner. Platelet exocytosis and aggregation induced by Cvx were inhibited by an anti-integrin alpha2beta1 monoclonal antibody (6F1) and by the Fab fragments of a polyclonal anti-glycoprotein VI (GPVI) antibody. Both the adhesion of platelets to Cvx and the Cvx-induced increase in [Ca2+]i were inhibited by anti-GPVI Fab fragments but not by 6F1. Ligand blotting assay showed that 125I-Cvx binds to a 57-kDa platelet protein with an electrophoretic mobility identical to that of GPVI. In addition, we observed the following: (i) 125I-Cvx binds to GPVI immunoprecipitated by the anti-GPVI antibody from a platelet lysate, and (ii) Cvx inhibits the binding of anti-GPVI IgG to GPVI. Taken together, these results demonstrate that GPVI behaves as a Cvx receptor and that the alpha2beta1 integrin appears to be involved in the later stages of Cvx-induced platelet activation, i.e. exocytosis and aggregation.

Antibodies, Monoclonal↗
PubMed1997

Molecular cloning and expression of bothrojaracin, a potent thrombin inhibitor from snake venom.

Bothrojaracin is a potent and selective thrombin inhibitor that has been isolated from the venom of Bothrops jararaca. It does not interact with the catalytic site of the enzyme but binds to both anion-binding exosites 1 and 2 resulting in a potent inhibition of thrombin activity towards fibrinogen and platelets [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C. & Bon, C. (1993) Biochemistry 32, 10794-108021. Bothrojaracin is a 27-kDa protein composed of two disulfide-linked polypeptide chains, A and B, of 15 kDa and 13 kDa, respectively. The sequences of A and B chains determined by molecular cloning exhibit a high degree of identity with other snake venom lectin-like proteins. In contrast to other ligands that interact with thrombin exosite 1, the amino acid sequence of bothrojaracin does not contain an acidic sequence similar to the C-terminal tail of hirudin. Expression of functional bothrojaracin was achieved in COS cells upon transfection with two pcDNA3 vectors containing the complete cDNAs. Recombinant bothrojaracin, which was secreted into the medium, was able to bind to and inhibit thrombin. When expressed alone, the B chain formed inactive dimers that were secreted into the culture medium. In contrast, no bothrojaracin-related protein was detected in conditioned media from cells transfected with the A chain.

Amino Acid Sequence↗
PubMed1997

Novel expression and localization of active thrombomodulin on the surface of mouse brain astrocytes.

Thrombin's potent effects on astrocytes are mediated by a specific receptor and inhibited by a serpin, protease nexin I (PNI). Thrombomodulin (TM), a membrane protein that forms complexes with thrombin, changing its enzymatic specificity, has not been studied in astrocytes. In primary astrocyte cultures, using Western blotting and immunocytochemistry, we found a 70 kDa TM band and TM localized to the surface with an anti-mouse TM monoclonal antibody. By reverse transcriptase coupled with polymerase chain reaction (RT-PCR), we found the correct sequence for mouse TM mRNA in astrocytes. Finally, we documented calcium-dependent activation of protein C by a thrombin:TM complex with thrombin added to the astrocytes. These results indicate the presence of functionally active TM at the astrocyte surface and add support to a role for thrombin signaling in the nervous system.

Animals↗
PubMed1996

Bothrojaracin: a potent two-site-directed thrombin inhibitor.

The thrombin inhibitor, bothrojaracin [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C., & Bon, C. (1993) Biochemistry 32, 10794-10802], is a 27 kDa protein isolated from the venom of Bothrops jararaca that blocks several thrombin functions, including fibrinogen clotting, platelet activation, and fibrin and thrombomodulin binding, but does not interact with the catalytic site. In the present report, we show that the high affinity binding of alpha-thrombin to immobilized bothrojaracin (Kd = 0.6 nM) is inhibited by the C-terminal peptide of hirudin and that the gamma-cleavage within exosite 1 reduces the affinity of bothrojaracin for thrombin (Kd = 0.3 microM), indicating that bothrojaracin binding to exosite 1 is a major determinant of the thrombin-bothrojaracin interaction. In addition, we show that bothrojaracin decreases the rate of inhibition of alpha- and gamma-thrombin by the antithrombin III-heparin complex. Competition of bothrojaracin with heparin or prothrombin fragment 2 for binding to thrombin indicates that bothrojaracin not only binds exosite 1 but also binds exosite 2 or in close proximity. Bothrojaracin binds to the thrombin precursor, prothrombin. This interaction is calcium-independent and is prevented by heparin, suggesting that it is mediated by exosite 2. Bothrojaracin inhibits platelet activation induced by clot-bound thrombin and slowly dissociates thrombin from the fibrin clots. Altogether, our results indicate that the high affinity of bothrojaracin for thrombin is supported by a double-site interaction and results in an efficient inhibition of both soluble and clot-bound thrombin.

Animals↗
PubMed1996

Myoblast fusion promotes the appearance of active protease nexin I on human muscle cell surfaces.

Protease nexin I (PNI) is a 43- to 50-kDa glycoprotein capable of inhibiting a number of serine proteases and belongs to the serpin superfamily. PNI is identical to glia-derived nexin, a neurite outgrowth promoter by virtue of its thrombin-inhibiting activity. Of particular relevance to neuromuscular biology and pathology, PNI was the first serpin shown to be highly localized to the neuromuscular junction and it maps to precisely the same locus as autosomal recessive amyotrophic lateral sclerosis (ALSJ) at chromosome 2q33-35. In the present report, we now show that in cultures of human skeletal muscle, PNI protein is expressed only after myoblast fusion into multinuclear myotubes and is localized in patches on their surfaces. We performed complex formation experiments with labeled thrombin, another target protease for PNI, with intact human muscle cells in culture. We detected specific SDS-stable PNI/thrombin complexes in myotube extracts only, indicating that active PNI was bound to their surfaces. We studied the gene expression of PNI mRNA using a 300-bp cDNA synthesized from the published sequence of human PNI. Confirming the protein data, upregulation of PNI appears in myotubes using Northern blot analysis. The current results reinforce the hypothesis that the regulation of the balance of serine proteases and serpins, such as PNI, is involved in muscle differentiation. They also prompt us to explore PNI abnormalities in several neuromuscular diseases, including ALSJ.

Amyloid beta-Protein Precursor↗
PubMed1996

Thrombin interaction with platelet membrane glycoprotein Ib.

Platelet activation by low doses of thrombin allows the amplification thrombin formation and thereby plays an important role in the development of thrombi. Although thrombin-induced platelet activation is elicited via the cleavage of its specific receptor (TR), platelet membrane glycoprotein Ib (GPIb) is required for responses to low concentrations of thrombin, as evidenced from the observation that GPIb-deficient platelets are characterized by a decreased sensitivity to thrombin and a low rate of activation. Glycoprotein Ib is an integral membrane protein composed of two disulfide-linked chains noncovalently associated to glycoproteins IX and V. As the receptor of the von Willebrand factor (vWF), GPIb plays a main role in platelet adhesion to the subendothelium. There are 25,000 copies of GPIb at the platelet surface but only a limited number of them appear to be involved in the high-affinity binding of thrombin. The catalytic site of thrombin is not involved in the interaction with GPIb. In contrast, competitive inhibition of GPIb-thrombin interaction by the C-terminal tail of hirudin, fibrin(ogen), and thrombomodulin indicates that thrombin exosite 1 is essential for GPIb binding. A hydrophylic domain located on the 45-kd N-terminal domain of GPIb alpha is involved in thrombin binding, and in particular, a stretch of negatively charged residues appears to make ionic interactions with thrombin. The same region of GPIb also contributes to the vWF binding site that should be very close to and even overlapping the thrombin-binding site. Despite GPIb and TR both interacting with thrombin exosite 1, the soluble fragment of GPIb does not modify the hydrolysis by thrombin of its target peptidic bond on TR, indicating that these two proteins bind to discrete subsites within exosite 1 and that the promoting effect of GPIb on TR-coupled responses depends on the anchorage of these proteins to the platelet membrane.

Amino Acid Sequence↗
PubMed1995

Role of the thrombin insertion loop 144-155. Study of thrombin mutations W148G, K154E and a thrombin-based synthetic peptide.

Thrombin is a multifunctional serine protease that plays a critical role in hemostasis. Crystallographic studies revealed that the insertion loop, residues 144-155 (human thrombin B chain numbering) located on the surface of thrombin, might be involved in the access of substrates to the active-site of the enzyme. This loop has also been proposed as a potential candidate for a binding site for thrombomodulin and selected thrombin substrates. In order to examine this hypothesis, we have introduced single amino acid substitutions into the loop 144-155 (W148G, K154E). These point mutations did not result in major changes in thrombin specificity. However, the mutant thrombins presented slight modifications in their catalytic activity on the tripeptidic substrate H-D-Lys-(epsilon-benzyloxycarbonyl)-Pro-Arg-NH-nitroanilide ([K154E]thrombin) or tosyl-Gly-Pro-Arg-NH-nitroanilide ([W148G]thrombin), and in the second-order rate constants of inhibition by antithrombin III ([K154E]thrombin) and ([W148G]thrombin) compared to recombinant wild-type thrombin. Kinetics of fibrinogen hydrolysis were minimally affected by the K154E mutation and were not affected by the W148G mutation. Neither of the mutations affected thrombin interaction with hirudin or its C-terminal tail, protein C activation by thrombin or thrombin-thrombomodulin, or platelet activation. We also examined the properties of a synthetic peptide corresponding to the sequence T147-S158. The synthetic peptide T147-S158 did not inhibit thrombin interaction with fibrin, thrombomodulin or protein C. Together, our results indicate that the thrombin loop 144-155 is indirectly involved in the catalytic function of the enzyme, most probably by limiting the access of the substrates to the catalytic site, and argue against the presence of a recognition exosite for fibrin(ogen), thrombomodulin or platelets within the loop.

Adult↗
PubMed1995

Thrombin interaction with a recombinant N-terminal extracellular domain of the thrombin receptor in an acellular system.

The cDNA of the human endothelial cell thrombin receptor has been cloned and a chimeric fusion protein consisting of glutathione-S-transferase (GST) and the portion 25-97 corresponding to the N-terminal first extracellular domain of the thrombin receptor (TRE) has been expressed in Escherichia coli. Introduction of a factor Xa cleavage site in the fusion protein allowed purification of TRE after removal from the GST carrier protein. Purified GST-TRE or TRE have been tested in solution for their ability to interact with thrombin. alpha-Thrombin cleaved the fusion protein at position Arg-41-Ser-42 of TRE in a time- and concentration-dependent manner and GST-TRE competed with the tripeptidic substrate S-2238 for hydrolysis by thrombin (Ki = 0.5 microM). gamma-Thrombin that lacks the anion-binding exosite was 100-fold less potent than alpha-thrombin at cleaving GST-TRE. TRE competed with polymerizing fibrin monomers for binding to thrombin (Ki = 7.5 microM). The cleavage of GST-TRE by alpha-thrombin was inhibited by several alpha-thrombin exosite ligands such as the C-terminal peptide of hirudin, thrombomodulin and fibrin(ogen) fragment E. In contrast, platelet glycocalicin did not inhibit GST-TRE cleavage. In conclusion, the use of purified soluble GST-TRE allowed us to derive an affinity constant for thrombin interaction with the N-terminal domain of the receptor and to confirm the location of the cleavage site at Arg41-Ser-42 of the receptor. The importance of the thrombin anion-binding exosite for thrombin receptor recognition is highlighted by the low reactivity of gamma-thrombin for GST-TRE and by competition experiments, which in addition indicate that binding sites for fibrin(ogen), thrombomodulin and GST-TRE are overlapping. In contrast, binding of thrombin to GST-TRE and glycocalicin are not mutually exclusive, indicating that glycocalicin and TRE interact with discrete subsites within the large groove that constitutes the anion-binding exosite.

Amides↗
PubMed1995

Thrombin specificity.

A model of thrombin interaction with distinct substrates or ligands has been derived from the crystallographic studies of thrombin-inhibitors complexes, and buttressed by functional studies with mutant thrombins, thrombin proteolytic derivatives or antibodies against thrombin. The unique specificity of thrombin for its substrates and ligands may be ascribed to multiple interactions with both the active site cleft and exosite(s) distinct from the active site. Two prominent insertion loops around Trp 50 and Trp 148 project over the active site cleft and play an important role in the substrates selection. Several substrates (fibrinogen, thrombin receptor, heparin cofactor II) or ligands (thrombomodulin, glycoprotein Ib) interact with a large exosite located on the surface of the loop segment 65-76, mainly constituted of basic amino acids, designated anion binding exosite 1. Interaction with these various macromolecules appears to involve a limited number of residues within the large exosite 1. It is conceivable that exosite 1 contains distinct subsites, although most of them may overlap. A second basic exosite (anion binding exosite 2) is located close to the carboxy-terminal B chain helix. Exosite 2 interacts with heparin, the chondroitin sulfate moiety of thrombomodulin and prothrombin activation fragment 2. Interaction of ligands with either exosite 1 or exosite 2 leads to conformational changes of the thrombin molecule, that may be important determinants of thrombin specificity. Whether exosite 2 cooperates with exosite 1 for thrombin interaction with fibrin(ogen) or the thrombin receptor remains to be determined.

Amino Acid Sequence↗
PubMed1994

An autoantibody directed against human thrombin anion-binding exosite in a patient with arterial thrombosis: effects on platelets, endothelial cells, and protein C activation.

An autoantibody, developed by a patient with severe and recurrent arterial thrombosis, was characterized to be directed against the anion-binding exosite of thrombin, and inhibited all thrombin interactions requiring this secondary binding site without interfering with the catalytic site. The effect of the antibody was studied on thrombin interactions with platelets and endothelial cells from human umbilical veins (HUVEC). The autoantibody specifically and concentration-dependently inhibited alpha-thrombin-induced platelet activation and prostacyclin (PGI2) synthesis from HUVEC. It had no effect when gamma-thrombin or the thrombin receptor activation peptide SFLLR were the inducers. The effect of the antibody on protein C activation has been studied. The antibody blocked the thrombin-thrombomodulin activation of protein C. The inhibition of the activation was maximal with a low concentration of thrombomodulin. The fact that the autoantibody inhibited concentration-dependent alpha-thrombin-induced platelet and endothelial cell functions emphasizes the crucial role of the anion-binding exosite of thrombin to activate its receptor. In regard to the pathology, the antibody inhibited two vascular processes implicated in thrombin-antithrombotic functions, PGI2 secretion, and protein C activation, which could be implicated in this arterial thrombotic disease.

Anions↗
PubMed1993

Bothrojaracin, a new thrombin inhibitor isolated from Bothrops jararaca venom: characterization and mechanism of thrombin inhibition.

A new thrombin inhibitor, bothrojaracin, has been identified and purified to homogeneity from the venom of Bothrops jararaca, the most common venomous snake of South America. Bothrojaracin has an isoelectric point of 4.2 and a molecular mass of 27 kDa and is made of two distinct polypeptide chains of 15 and 13 kDa, linked by disulfide bridges. Purified bothrojaracin is devoid of phospholipase A2, amidolytic, or fibrino (geno)lytic activity. Bothrojaracin forms a noncovalent complex with alpha-thrombin, without changing its catalytic activity on small peptide substrates. Bothrojaracin behaves as a potent and specific antagonist of thrombin-induced platelet aggregation and secretion, characterized by an IC50 ranging from 1 to 20 nM depending on the alpha-thrombin concentration. Bothrojaracin prolongs fibrinogen clotting time, and this effect is related to a competitive inhibition of the binding of alpha-thrombin to fibrin(ogen) (Ki 15 nM). Binding of alpha-thrombin to thrombomodulin is inhibited up to 87% by bothrojaracin, and the rate of protein C activation by alpha-thrombin is also decreased. Bothrojaracin antagonizes the inhibition of thrombin amidolytic activity by hirudin. These results indicate that bothrojaracin acts as a very potent ligand of the exosite of alpha-thrombin.

Amino Acid Sequence↗
PubMed1993

Late-fibrin(ogen) fragment E modulates human alpha-thrombin specificity.

Fibrinogen contains at least two independent sites having demonstrable affinity for alpha-thrombin. One of these two sites, located in the fibrin E domain, binds to structures within the anion-binding exosite of alpha-thrombin. Taking advantage of its solubility, we have used late-fibrin(ogen) fragment E in competition experiments to examine its effect on alpha-thrombin specificity. We show that fragment E modulates alpha-thrombin enzymic activity towards small synthetic substrates, suggesting that fibrin-thrombin interaction might induce subtle changes in the conformation near the catalytic center of the enzyme. In addition, fragment E behaved as a competitive inhibitor of alpha-thrombin-catalyzed fibrinopeptide-A cleavage (Ki = 5.2 +/- 1.3 microM), indicating that alpha-thrombin interaction with the fibrin moiety of fibrinogen makes a major contribution to the efficacy of fibrinogen hydrolysis. Fragment E inhibited alpha-thrombin-induced serotonin release by platelets (concentration required to obtain 50% inhibition, IC50 = 10 microM) and alpha-thrombin binding to GPIb. Fragment E competitively inhibited alpha-thrombin binding to thrombomodulin (Ki = 18.3 +/- 0.8 microM) but did not inhibit protein-C activation in the absence of thrombomodulin. The data are consistent with the proposal that fibrin, platelet GPIb and thrombomodulin bind to overlapping, but probably non-identical sites, while protein C binds to an independent site on alpha-thrombin.

Amino Acid Sequence↗
PubMed1993

Enhancement of the synthesis and secretion of nerve growth factor in primary cultures of glial cells by proteases: a possible involvement of thrombin.

Newborn rat brain astrocytes cultured in vitro in a chemically defined medium are shown to secrete enhanced levels of nerve growth factor (NGF) when they are exposed to various types of proteases. Proteolytic enzymes such as alpha-thrombin or collagenase induce a continuous, dose-dependent enhancement of the levels of cell-secreted NGF. Incubation of astrocytes for a 24-h period with 300 ng/ml of alpha-thrombin (approximately 9 nM, or 1 U/ml) results in an increase of the levels of cell-secreted NGF by a factor of three- to fourfold, and at doses 10 times higher, stimulation by a factor of up to four- to fivefold was observed. This phenomenon reflects an enhancement of the cellular pool of NGF mRNA, already noticeable after 3 h of treatment, which is preceded by a temporary activation of protooncogenes encoding transcription factors of the AP-1 family, such as c-fos, c-jun or junB. Trypsin, plasmin, alpha-chymotrypsin, or elastase also enhanced, to different extents, the levels of cell-secreted NGF. However, unlike alpha-thrombin or collagenase, these enzymes cause, above a critical concentration, an extensive cell detachment from the solid support, and this is accompanied by a decrease of their activity on the production of NGF, so that their dose-response curves are bell shaped. Stimulation was maximal at those concentrations that cause a limited loosening of the cell-substratum interactions, as evidenced by a retraction of some cell processes after 24 h of treatment. Studies of the effect of alpha-thrombin indicate that the proteolytic activity itself is required to enhance the production of NGF by astrocytes. Inactivation of alpha-thrombin with D-phenyl-alanyl-L-propyl-L-arginine chloromethyl ketone, phenylmethylsulfonyl fluoride, antithrombin III, or hirudin results in a marked decrease of the stimulatory effect. Furthermore, the prolonged presence of alpha-thrombin is required to elicit a maximal effect on the levels of extracellular NGF, which was observed after 48 h of treatment. It is known that some effects of alpha-thrombin require binding to the cell surface. We found that gamma-thrombin, which still has some proteolytic activity but has lost its ability to bind to the cell surface, is almost as potent as alpha-thrombin in promoting the release of NGF. It is concluded that the effect of thrombin on NGF synthesis is essentially mediated by its proteolytic activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Thrombin interaction with platelet glycoprotein Ib: effect of glycocalicin on thrombin specificity.

We describe here the alteration of thrombin specificity induced by its interaction with glycocalicin. Glycocalicin is the external part of platelet glycoprotein Ib alpha (GPIb alpha) and contains binding sites for von Willebrand factor and thrombin. Taking advantage of its solubility, we have used glycocalicin in competition assays on various thrombin activities. Glycocalicin did not inhibit chromogenic substrate hydrolysis nor diisopropylfluorophosphate iPr2 (PF) incorporation, indicating that thrombin binding to GPIb does not alter access to or the conformation of the thrombin catalytic site. Glycocalicin competitively inhibited thrombin binding to fibrin (Ki = 0.1 mumol/L) and blocked fibrinogen clotting activity of thrombin. Glycocalicin also inhibited thrombin binding to thrombomodulin in a competitive manner (Ki = 3 to 5 mumol/L), but failed to prevent thrombin interaction with protein C in the absence of thrombomodulin. Previous results have indicated that GPIb binds to thrombin within the anion binding exosite masked by the carboxy-terminal hirudin peptide 54-65. The present results confirm the implication of the anion binding exosite in GPIb recognition, and further indicate that the thrombin binding site for GPIb overlaps with the thrombin binding sites for fibrin and thrombomodulin, whereas it is distinct from the thrombin binding site for protein C. Some of the structural requirements for thrombin binding to GPIb appear to be very similar to those reported for binding to its platelet receptor. However, thrombin-GPIb interaction does not appear to compete with receptor hydrolysis but rather increases the sensitivity and the rate of platelet responses elicited by the receptor.

Amino Acid Sequence↗
PubMed1991

Effect of the hirudin carboxy-terminal peptide 54-65 on the interaction of thrombin with platelets.

The carboxy-terminal region of hirudin (residues 54-65) has previously been shown to inhibit thrombin clotting activity without binding to the catalytic site of the enzyme. In the present study, the effect of hirudin 54-65 on thrombin interaction with specified platelet proteins has been investigated. Hirudin 54-65 was found to inhibit thrombin-induced platelet aggregation and secretion in a dose-dependent manner. Substitution of either Phe56, Glu57, Ile59, Pro60 or Leu64 showed that these residues were critical for inhibition of thrombin-induced platelet activation whereas sulfation of Tyr63 increased the inhibitory potency of the peptide. Hydrolysis of glycoprotein V, a platelet membrane substrate for thrombin, was only partially inhibited by hirudin 54-65. Although hirudin 54-65 did not decrease the amount of thrombin bound to platelets during cross-linking experiments, it was found to inhibit the specific binding of thrombin to platelet glycoprotein Ib. Since the carboxy-terminal region of hirudin has previously been reported to bind near the trypsin-catalyzed beta cleavage site, we have analyzed the consequences of alpha to beta-thrombin conversion on both thrombin-hirudin 54-65 interaction and thrombin activity toward platelets. The beta cleavage induced a decrease in the affinity of thrombin for both glycoprotein Ib and hirudin 54-65. Altogether, our results indicate that thrombin recognition sites for hirudin 54-65 and platelet membrane glycoprotein Ib share common structures located near the beta cleavage site at Arg 73 on the thrombin B chain.

Amino Acid Sequence↗
PubMed1991

Effect of phosphopyridoxylation on thrombin interaction with platelet glycoprotein Ib.

The purpose of this study was to determine the effect of chemical modification of lysyl residues on thrombin interaction with platelet membrane proteins. Modification of lysyl residues by pyridoxal-5'-phosphate affected two different sites on thrombin and resulted in a greatly decreased binding to platelets. Using a crosslinking bifunctional reagent [bis(sulphosuccinimidyl) suberate (BS3)], we show that modified thrombin retained the ability to form high molecular mass (greater than or equal to 400 kDa) complexes with yet unidentified platelet proteins and to bind to platelet protease nexin I, but had lost the ability to bind to platelet glycoprotein Ib (GPIb). As previously reported by others, heparin protected one of the two sites from phosphopyridoxylation. In contrast modified thrombin, heparin-protected modified thrombin retained the ability to bind to GPIb, indicating that the lysyl residue(s) protected by heparin from the modification are essential for GPIb binding. While unprotected modified thrombin failed to bind hirudin, heparin-protected modified thrombin retained its ability to bind the carboxy-terminal hirudin peptide H54-65. Tritium-labelling of the modified lysyl residues and degradation of modified thrombins by CNBr or trypsin confirmed that the lysyl residue(s) protected by heparin and essential for GPIb binding are located in the thrombin binding domain for the carboxyl-terminal tail of hirudin, within the sequence 18-73 of the thrombin B chain.

Binding Sites↗
PubMed1990

The common pathway for alpha- and gamma-thrombin-induced platelet activation is independent of GPIb: a study of Bernard-Soulier platelets.

The responses to alpha- and gamma-thrombin were studied in normal and Bernard-Soulier platelets labelled with [32P]phosphate, to investigate the relationship between thrombin binding to the platelet membrane glycoprotein Ib (GPIb) and thrombin-induced platelet activation. For this purpose we conducted parallel studies of the kinetics of platelet aggregation, granule secretion, hydrolysis of polyphosphoinositides, formation of phosphatidic acid, phosphorylation of the myosin light chain (p20) and of the 43 kDa protein (p43), and thromboxane B2 formation. Like alpha-thrombin, gamma-thrombin activated control platelets via all the above metabolic responses, but only after a prolonged lag. In Bernard-Soulier platelets, alpha-thrombin induced polyphosphoinositide hydrolysis and phosphatidic acid formation, p20 and p43 phosphorylation, thromboxane B2 formation, secretion and to a lesser extent aggregation, but only after a prolonged lag. The metabolic responses of Bernard-Soulier platelets to gamma-thrombin were very similar to those of control platelets. We have previously showed that GPIb which is not present in Bernard-Soulier platelets binds alpha- but not gamma-thrombin. The present results indicate that thrombin binding to GPIb is not directly coupled either with the activation of phospholipase C specific to polyphosphoinositides, or with the activation of protein kinase C and phospholipase A2. However, thrombin binding to GPIb appears to promote an early mechanism which accelerates all the platelet responses.

Adult↗