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Biomedical subjects

R Landolfi

Publications and source records attributed to R Landolfi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Effect of high- and low-molecular-weight heparins on thrombin-thrombomodulin interaction and protein C activation.

BACKGROUND: Thrombin-thrombomodulin (TM) interaction, which is critical for accelerating the protein C anticoagulant pathway, involves the heparin-like domain of TM. This study was aimed at investigating the possible effect of heparin on thrombin-TM binding and protein C activation. METHODS AND RESULTS: The affinity of thrombin-TM interaction was studied by a functional method, based on the ability of thrombin-TM adduct to activate protein C, and by evaluation of the binding of thrombin to immobilized TM. Both experimental approaches showed that the affinity of thrombin-TM interaction was decreased by micromolar heparin concentrations. Heparin had no significant effect when a recombinant TM form, lacking the chondroitin sulfate moiety, was used. Furthermore, it was also shown that the inhibitory effect of heparin was directly proportional to the heparin molecular mass (molecular weight range, 3 to 16 kDa), which suggests that the effect was mediated by formation of electrostatic bonds between heparin and thrombin. CONCLUSIONS: These results indicate that heparin at therapeutic concentrations reduces the affinity of thrombin for TM and the rate of protein C activation. The magnitude of this effect is proportionally linked to the molecular mass of heparin.

Anticoagulants↗
PubMed1998

Binding of human alpha-thrombin to platelet GpIb: energetics and functional effects.

Thrombin interaction with platelet glycocalicin (GC), the 140 kDa extracytoplasmic fragment of the membrane glycoprotein Ib, was investigated by using a solid-phase assay. Thrombin bound to GC-coated polystyrene wells was detected by measuring the hydrolysis of a chromogenic substrate. The monoclonal antibody LJ-Ib10, which specifically binds to the thrombin-binding site of GC, could displace thrombin from immobilized GC, whereas the monoclonal antibody LJ-Ib1, which interacts with the von Willebrand factor-binding domain of GC, did not affect thrombin binding to GC. Competitive inhibition of thrombin binding to immobilized GC was also observed using GC in solution or ligands that bind to the thrombin heparin-binding site, such as heparin and prothrombin fragment 2. Furthermore functional experiments demonstrated that GC binding to thrombin competes with heparin for thrombin inactivation by the antithrombin III-heparin complex as well. Thrombin-GC interaction was also studied as a function of temperature over the range 4-37 degreesC. A large negative heat capacity change (DeltaCp), of -4.14+/-0.8 kJ.mol-1.K-1, was demonstrated to dominate the thermodynamics of thrombin-GC complex-formation. Finally it was demonstrated that GC binding to thrombin can allosterically decrease the enzyme affinity for hirudin via a simultaneous decrease in association rate and increase in the dissociation velocity of the enzyme-inhibitor adduct. Together these observations indicate the GC binding to the heparin-binding domain of thrombin is largely driven by a hydrophobic effect and that such interaction can protect the enzyme from inhibition by the heparin-anti-thrombin III complex.

Antithrombin III↗
PubMed1998

Measurement of plasma fibrinogen concentration by the prothrombin-time-derived method: applicability and limitations.

A prothrombin-time-derived method was used to measure plasma fibrinogen concentration (PFC) in 286 samples from 242 normal and 44 orally anticoagulated subjects. Absorbance changes at 405 nm (deltaOD) during the clotting process were obtained by an automatic coagulometer and their relationship with plasma fibrinogen concentration (range 90-1090 mg/dl), measured by the Clauss method, was investigated. A weighted linear regression between the deltaOD and the Clauss-derived PFC values provided the best fit of the experimental data. The fitting equation was found to be reliable and accurate for PFC determination in normal subjects, whereas a systematic overestimate of fibrinogen level was demonstrated in plasma with high fibrinogen concentrations (> 400 mg/dl) and in plasma from anticoagulated patients. The systematic overestimate in the latter samples could be a result of an increased fibrin gel turbidity, as shown by in-vitro experiments using purified fibrinogen clotted by different thrombin concentrations. The PFC overestimate by the prothrombin-time-derived method could also be experimentally reproduced by competitively inhibiting thrombin-fibrinogen interaction by hirudin 54-65 peptide and the fibrinogen fragment E. A similar qualitative result was also found for the prothrombin-time-derived method in the presence of the Gly-Pro-Arg-Pro peptide, which competitively inhibits the end-to-end fibrin aggregation process. Notably, under both the above experimental conditions, the Clauss method underestimated the PFC. On the other hand, the 'clot recovery' method was minimally affected by the above inhibitors. These results indicate that the prothrombin-time-derived method is accurate and precise for most routine purposes. Its precision seems inadequate, however, under those conditions where the prothrombin time is prolonged (such as anticoagulant therapy) and in the presence of high fibrinogen levels.

Anticoagulants↗
PubMed1998

Bleeding and thrombosis in myeloproliferative disorders.

Patients with polycythemia vera and essential thrombocythemia have a hemostatic imbalance, the mechanisms of which are still elusive. Hemorrhagic tendency usually manifests in patients with high platelet count and reveals the clinical and laboratory features of acquired von Willebrand's disease. In most patients, however, the increased risk of arterial and venous thromboses constitutes the main therapeutic challenge. Data from large retrospective studies have recently allowed us to reassess the clinical epidemiology of these manifestations. Erythromelalgia and other peculiar microcirculatory disturbances are selectively sensitive to aspirin. This finding, and the thromboxane A2 hyperproduction existing in polycythemic and thrombocythemic subjects, provide the rationale for the use of low-dose aspirin in these patients. The efficacy and safety of this treatment in patients with polycythemia vera is being tested in a large scale, randomized trial. Appropriately designed clinical studies are also needed for defining the neoplastic risk of commonly used chemotherapic agents and the antithrombotic efficacy of alternative cytoreductive strategies.

Hemorrhage↗
PubMed1998

Increased thromboxane metabolites excretion in liver cirrhosis.

An augmented systemic production of thromboxane (TX) A2, as assessed by urinary excretion of the thromboxane metabolites, has been described in severe liver cirrhosis. However, the significance of this finding remains unclear since in liver cirrhosis a number of phenomena i.e. altered hepatic TXA2 metabolism, increased intrasplenic platelet destruction, may affect TXA2 entry into systemic circulation as well as its metabolism. In order to further clarify this, we measured both major enzymatic metabolites of TXB2 in the urine of 44 patients affected by liver cirrhosis, subdivided in three classes on the basis of Child-Pugh criteria. Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were assayed with previously validated RIA techniques. The urinary excretion rate of 11-dehydro-TXB2 was significantly (p = 0.0001) increased in the cirrhotic patients (673.5 pg/mg cr, median) in comparison with the controls (275 pg/mg cr, median) but no significant difference could be demonstrated among the excretion rates of the three patient subgroups. The excretion rate of 2,3 dinor-TXB2 was also significantly (p = 0.0001) increased in the patients (824 pg/mg cr, median) in comparison with controls (175 pg/mg cr, median), with a significant (p < 0.05) increase from class A (381 pg/mg cr) to class C (1337 pg/mg cr). The sum of the two enzymatic metabolites was significantly (p = 0.0001 ) increased in the cirrhotic patients in comparison to controls, with a progressive increase from class A (1003 pg/mg cr, median) to class C (2240 pg/mg cr, median). The urinary excretion of 2,3 dinor-TXB2 was significantly (p = 0.008) related to plasma prothrombin fragment 1+2 (F1+2). This study provides further evidence of increased thromboxane biosynthesis in liver cirrhosis. Moreover, we demonstrate intraliver shift of thromboxane metabolic disposition, due to progressive liver decompensation, because only the fraction undergoing beta-oxidation to 2,3-dinor-TXB2 was progressively increased with the degree of liver failure. We, also, find a significant correlation between urinary excretion of 2,3-dinor-TXB2 and plasma F1+2, suggesting that clotting activation could partly account for in vivo platelet activation.

Adult↗
PubMed1997

Allelotype analysis in the evolution of chronic myelocytic leukemia.

To elucidate the genetic events that may play important roles in the progression of chronic myelocytic leukemia (CML), we performed allelotype analysis in 30 patients with CML as the disease transformed to accelerated phase or blast crisis (21 myeloid and 9 lymphoid cases). DNAs were extracted from slides of bone marrow smears or from freshly isolated bone marrow mononuclear cells. The DNAs from the same individuals in both chronic phase and either blast crisis or accelerated phase were analyzed at 82 microsatellite markers, which mapped to each of the autosomal arms except the short arms of the acrocentric chromosomes. Loss of heterozygosity (LOH) on at least one locus was observed in 21 of the 30 cases (70%) as the disease progressed. Frequent allelic loss of > or = 20% of the informative cases was observed on chromosome arms 1p (35%), 7p (21%), 19p (20%), and 20q (29%). Allelic losses were also analyzed according to phenotypes. LOH of > or = 20% was detected on 1p (29%), 18p (20%), and 20q (27%) in myeloid blast crisis, and on 1p (50%), 4p (25%), 7p (43%), 9p (29%), 18q (25%), 19p (43%), and 20q (33%) in lymphoid blast crisis. Serial cytogenetic information was available for most of our cases with LOH on these arms, and only one case had loss of both chromosomes 9 and 20. Fractional allelic loss, calculated for each sample as the total number of chromosomal arms lost/total number of arms with information, showed a median value of 0.06 and a mean of 0.098 (range 0 to 0.60). These results suggest that tumor suppressor genes especially on 1p, 7p, 19p, and 20q probably have an important role in the progression to blast crisis of CML.

Adult↗
PubMed1997

European Collaboration on Low-dose Aspirin in Polycythemia Vera (ECLAP): a randomized trial.

Thrombotic complications characterize the clinical course of polycythemia vera (PV) and represent the main cause of morbidity and mortality. However, uncertainty still exists as to the benefit/risk ratio of aspirin prophylaxis in this setting. In vivo platelet biosynthesis of thromboxane A2 is enhanced and can be suppressed by low-dose aspirin in PV, thus providing a rationale for assessing the efficacy and safety of a low-dose aspirin regimen in these patients. The Gruppo Italiano Studio Policitemia Vera has recently performed a pilot study on 112 patients randomized to receive aspirin, 40 mg daily, or placebo and followed for 16 +/- 6 months (mean +/- SD). This study showed that low-dose aspirin is well tolerated in PV patients, and that a large-scale efficacy trial is feasible in this setting. In this article we report the protocol of the European Collaboration on Low-dose Aspirin in Polycythemia Vera (ECLAP) study, which is a randomized trial designed to assess the risk/benefit ratio of low-dose aspirin in PV. To estimate the size and the follow-up duration required for the ECLAP trial, a retrospective analysis of the clinical epidemiology of a large PV population has recently been completed by the Gruppo Italiano Studio Policitemia Vera. On this basis, approximately 3500 patients will be enrolled in the ECLAP study with a follow-up of 3 to 4 years. The uncertainty principle will be used as the main eligibility criterion: Polycythemic patients of any age, having no clear indication for or contraindication to aspirin treatment, will be randomized in a double-blind fashion to receive oral aspirin (100 mg daily) or placebo. According to current therapeutic recommendations, the basic treatment of randomized patients should be aimed at maintaining the hematocrit value < or = 45% in subjects aged < or = 50, and hematocrit < 45% as well as platelet count < 400 x 10(9)/L in patients aged > 50. Randomization will be stratified by participating center. The study is funded by the European Union BIOMED 2 program.

Aspirin↗
PubMed1997

Compound heterozygosity for two novel missense mutations in the prothrombin gene in a patient with a severe bleeding tendency.

The abnormal prothrombin gene of an Italian patient with a severe bleeding tendency and hypoprothrombinemia was selected for study and compared with the prothrombin genes of healthy controls. All the coding and their flanking regions and the 5'- and 3'-UT regions of the prothrombin gene were screened by analyzing the nucleotide sequence of the corresponding PCR products. The patient was found to be heterozygous for two novel point mutations: one at nucleotide 4251 in exon 6, which changes the codon for cysteine-138 (TGC) in the kringle 1 domain to that for tyrosine (TAC), and one at nucleotide 8812 in exon 10, which results in the replacement of tryptophan-357 (TGG) by cysteine (TGT) in the catalytic domain. Her mother was heterozygous for the Cys-138 Tyr mutation and her father heterozygous for the Trp-357 Cys mutation. Several other sequence variations were identified in the prothrombin genes from control individuals. Only the variations at nucleotide 4203 and 10253 could be established as polymorphisms.

Blood Coagulation Factors↗
PubMed1997

Thrombin interaction with platelet GpIB: role of the heparin binding domain.

The platelet membrane glycoprotein Ib (GpIb) has a high affinity binding site for alpha-thrombin whose occupancy is thought to positively modulate the thrombin-induced platelet activation. In this study, aimed at further characterizing the thrombin-GpIb interaction, two thrombin anion exosites referred to as "heparin binding site" (HBS) and "fibrinogen recognition site" (FRS) were investigated as the possible domains involved in GpIb binding. The role of thrombin HBS was explored by performing binding measurements of 125I-alpha-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of GpIb, in the presence of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studies showed that a) thrombin binding to GC could be competitively inhibited by heparin and b) the equilibrium association constant for thrombin-GC interaction was reduced up to ten-fold by chemical modifications at the HBS. On the other hand, the role of FRS in the thrombin-GC interaction could be excluded by other experiments showing that GC in solution could not influence the interaction of alpha-thrombin with two substrates which bind to both the catalytic site and the fibrinogen recognition site: 1) the thrombin receptor peptide 38-60 (TR, L38-E60) and 2) the A alpha-chain of fibrinogen. Altogether these results demonstrated that GC interaction with thrombin involves the enzyme heparin binding site, whereas the fibrinogen recognition site does not play a significant role.

Binding Sites↗
PubMed1997

Mechanisms of bleeding and thrombosis in myeloproliferative disorders.

Arterial and venous thromboses and microcirculatory disturbances such as erythromelalgia and neurologic and visual symptoms are the thrombotic manifestations occurring in Polycythaemia Vera and Essential Thrombocythaemia. The increased in vivo thromboxane A2 generation existing in these patients and the selective sensitivity of erythromelalgia to aspirin suggest that platelet PGG/H synthase products may be involved in transducing the increased thrombotic risk. The relationship between Thromboxane A2 production and thrombotic accidents will be investigated by the European Collaboration on Low-Dose Aspirin in Polycythaemia Vera (ECLAP) which will test the efficacy of low-dose aspirin by a randomised clinical trial. The haemorrhagic diathesis of polycythaemic and thrombocythaemic subjects is generally mild and spontaneous bleeding usually manifests in patients with very high platelet count. Its mechanism may be related to quantitative as well as to qualitative platelet changes. Possible mechanisms linking the high grade thrombocytosis to bleeding are consumption of von Willebrand factor and clot fragility due to a mechanical effect of the high platelet count or to inhibition of fibrin polymerization by platelet Glycoprotein Ib.

Blood Platelets↗
PubMed1996

Wiskott-Aldrich syndrome: report of an autosomal dominant variant.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder originally described as a clinical triad of thrombocytopenia with small platelets, eczema, and immunodeficiency. Impaired CD43 glycoprotein expression on lymphocytes is a typical hallmark of this disorder. The CD43 gene is located on chromosome 16, and the WAS gene, WASP, was recently isolated from the chromosome X p11.22-p11.23. This gene, mutated in WAS patients, encodes a protein that is likely to play a role in controlling the expression of CD43. However, the molecular mechanism(s) causing WAS are not yet known. Herein, we describe a three-generation family in which clinical and laboratory WAS features were expressed in six of nine subjects available for study. At variance with classic X-linked WAS, this disorder was characterized by the presence of thrombocytopenia with a broad spectrum of platelet size, including giant platelets, and was inherited as an autosomal dominant trait. This last finding led us to hypothesize a mutation of the CD43 gene. However, Southern blot analysis failed to detect structural abnormalities of this gene, and genotype analysis ruled out the possibility that a CD43 allele might be shared by the affected individuals. These findings indicate that an alteration(s) of an autosomal gene distinct from the CD43 gene is responsible for the disease. Thus, results from this family, providing the first observation of an autosomally transmitted WAS variant, indicate that genetic mechanism(s) leading to WAS are more complex than previously recognized.

Adolescent↗
PubMed1996

Effect of sodium on the energetics of thrombin-thrombomodulin interaction and its relevance for protein C hydrolysis.

Measurements of the apparent affinity constant for thrombomodulin (TM) binding to human alpha-thrombin as a function of both NA+ and temperature at constant ionic strength (0.15 M) showed that TM affinity increases in the presence of Na+ and vice versa. Moreover, this experimental strategy allowed us to accurately split the free energy of sodium binding into its entropic and enthalpic components for both the TM-free and TM-bound enzyme. Namely, at 25 degrees C, the value of delta G of sodium binding was found equal to -2.4 kcal/mol in the absence of TM and -3.6 kcal/mol for the thrombin-TM complex. The enthalpic contribution to the free energy of sodium binding is equal to -27 kcal/mol and -21 kcal/mol in the TM-free and TM-bound thrombin forms, respectively. Finally, the entropy change for sodium binding was also affected by TM, being equal to -83 cal/(mol deg) and -58 cal/(mol deg) in TM-free and TM-bound thrombin species, respectively. Moreover, the thermodynamic parameters for TM binding to Na+-free thrombin species were solved. TM binding is characterized by an enthalpy and entropy change equal to -10 kcal/mol and 2 cal/(mol deg), respectively, for Na+-free thrombin. It is well known that Na+ binding to thrombin causes conformational transitions and functional activation of the enzyme molecule. The finding that binding of thrombomodulin enhances thrombin affinity for sodium and vice versa raises the question as to whether the change of Na+ ligation induced by TM binding could contribute to the change in thrombin specificity for the hydrolysis of Protein C. Therefore, the effect of sodium binding to thrombin on the hydrolysis of human Protein C was extensively investigated. At both 25 and 37 degrees C the value of kcat/Km for Protein C hydrolysis by thrombin in the absence of TM was found to be enhanced by Na+ over a concentration ranging from 0 to 150 mM. Application of thermodynamic principles demonstrated that the Na+-thrombomodulin linkage contributes, under physiological conditions of sodium activity and temperature, to reduce significantly the transition-state stabilization free energy for Protein C hydrolysis.

Amino Acid Sequence↗
PubMed1996

Aspirin in polycythemia vera and essential thrombocythemia: current facts and perspectives.

The role of aspirin in the antithrombotic strategy of patients with polycythemia vera (PV) and essential thrombocythemia (ET) is highly controversial. Long considered unsafe on the basis of a single clinical trial testing very high doses in PV patients, aspirin is being increasingly used at lower dosage. The rationale for the use of aspirin in patients with PV and ET is provided by the efficacy of this agent in the treatment of microcirculatory disturbances of thrombocythemic states associated with myeloproliferative disorders and by recent evidence that asymptomatic PV and ET patients have persistently increased thromboxane (TX) A2-biosynthesis. This increase, which most likely reflects enhanced platelet activation in vivo, is independent of the platelet mass and blood viscosity and largely supressed by a short term low-dose aspirin regimen (50 mg/day for 7 days). Since enhanced TXA2 biosynthesis may play a role in transducing the increased thrombotic risk associated with PV and ET, long-term low-dose aspirin administration has been proposed as a possible antithombotic strategy in these subjects. The safety of this treatment in PV patients has been recently reassessed by the Gruppo Italiano per lo Studio della Policitemia Vera (GISP) which has followed for over one year 112 patients randomized to receive 40 mg/day aspirin or placebo. In the same study, serum TXB2 measurements provided evidence that the low-dose aspirin regimen tested was fully effective in inhibiting platelet cyclooxygenase activity. On this basis, a large scale trial aimed at assessing the antithrombotic efficacy of this approach is currently being organized. In patients with ET both the minimal aspirin dose required for complete inhibition of platelet cyclooxygenase and the safety of long-term aspirin administration need to be established prior to extensive clinical evaluation of this strategy.

Adult↗
PubMed1996

Feasibility of randomised clinical trials in rare diseases: the case of polycythemia vera.

Although it has long been recognised as the only reliable instrument for producing scientific evidence on the benefit/risk profile of therapeutic interventions, the technology of randomised clinical trials (RCT) is far from being the backbone of medical knowledge. Randomised clinical trials in polycythemia vera have been carried out when their methodology was being built up and, therefore, was rather unsatisfactory. Now we have aggressive cytoreductive treatments with chemotherapeutic agents loaded with doubts on long-term safety, while phlebotomy and preventive antiplatelet therapy are left to personal preferences because of debatable results of old, low-power clinical trials. A complex profile of uncertainties requires a simple, but articulated strategy of care and research to allow at the same time a reasonable transfer of the best available validated knowledge and a timely investigation of the most relevant questions. Without doubts, multi-country, collaborative RCTs is the key (not isolated or abstract) element of the current scenario. The declared background hypothesis is the willingness of a medical caring community of being, at the same time and with the same patients, a research community. The trial design comes in as the simplest technical way to deal with uncertainty. Data to be collected, criteria, contents, intensity of follow-up, and documentation of the events are exactly the same as those which are planned and adopted in routine care. One of the greatest achievements of the multicenter trials with this orientation has been to produce a "core" of data and practices, on which the main analyses will be made, but which at the same time reflect an optimal level of assistance to the majority of patients. The purpose of this paper is therefore twofold: a) to provide a brief methodological review of the controlled evidence available for the direction of therapeutic practice for PV: b) to outline and discuss the opportunity, general design, and feasibility of research strategies where a comparative large-scale trial between therapeutic alternatives could play a central role.

Cohort Studies↗
PubMed1995

Thrombin-thrombomodulin interaction: energetics and potential role of water as an allosteric effector.

The interaction of rabbit lung thrombomodulin (TM) and C-terminal hirudin 54-65 fragment (Hir54-65) with human alpha-thrombin were investigated by exploiting their competitive inhibition of thrombin-fibrinogen interaction. Measurements of Ki values for TM and Hir54-65 interactions with human alpha-thrombin performed over a temperature range spanning from 10 to 40 degrees C showed a constant enthalpy for both ligands. The enthalpic and entropic contributions to the free energy of binding, however, are different for TM and the hirudin peptide. The calculated values of delta H and delta S, in fact, were -47.3 +/- 2.51 kJ (-11.3 +/- 0.6 kcal)/mol and -42.7 +/- 7.9 J (-10.2 +/- 1.9 cal)/mol.K for the hirudin peptide, while being -22.9 +/- 2.09 kJ (-5.47 +/- 0.5 kcal)/mol and 102.50 +/- 6.69 J (24.5 +/- 1.6 cal)/mol.K respectively for TM binding. These findings indicate that the interaction between thrombin and Hir54-65 is largely driven by the enthalpic contribution, whereas the positive entropy change is the driving force for the formation of the thrombin-TM complex. In other experiments performed in the presence of various concentrations of either sorbitol or sucrose it could be demonstrated that the value of the equilibrium association constant for thrombin-TM interaction increases as a function of the osmotic pressure, while the thrombin-Hir54-65 interaction was not affected by the same conditions. Moreover, control experiments showed that no major conformational changes are produced on TM by osmotic pressures used in the present study. From these experiments it was calculated that roughly 35 water molecules are released into the bulk water upon TM binding. Such a phenomenon, which is likely to be responsible for the entropic change described above, indicates the relevance of hydration processes for the formation of the thrombin-TM adduct.

Allosteric Regulation↗
PubMed1995

Conformational transitions linked to active site ligation in human thrombin: effect on the interaction with fibrinogen and the cleavable platelet receptor.

An experimental strategy based on solution viscosity perturbation allowed us to study the energetics of amide-substrates, p-aminobenzamidine (p-ABZ) and proflavin binding to the catalytic site of two proteolyzed forms of alpha-thrombin, i.e. zeta- and gamma T-thrombin. These thrombin derivatives are cleaved at the Leu144-Gly150 loop and at the fibrinogen recognition exosite (FRS), respectively. A phenomenological analysis of thermodynamic data showed that the amide substrates and p-ABZ interactions with zeta-thrombin were respectively, associated with a chemical compensation (i.e. the linear relationship between entropy and enthalpy of binding) and a hydrophobic phenomenon (i.e. a change in the standard heat capacity). The latter was slightly lower than that previously observed for a alpha-thrombin (0.78 +/- 0.25 versus 1.01 +/- 0.17 kcal/mol K). Both phenomenon were absent in gamma T-thrombin. The interaction of a alpha-, zeta- and gamma T-thrombin with macromolecular substrates that "bridge-bind" to both the catalytic site (CS) and fibrinogen recognition exosite (FRS), such as fibrinogen and the cleavable platelet receptor (CPR), was also evaluated. These interactions were studied by following fibrinopeptide A (FpA) release and by measuring intraplatelet Ca2+ changes induced by thrombin-CPR interaction. It was found that the free energy of activation (RT ln Kcat/Km) for both fibrinogen and CPR hydrolysis followed the same hierarchy, i.e. alpha > zeta > gamma. Moreover, the values of delta Cp for alpha-, zeta- and gamma T-thrombin interaction with p-ABZ were found to be linearly correlated to the free energy of activation for both fibrinogen and CPR cleavage. In conclusion, these data demonstrate that: (1) the Leu144-Gly150 loop and the FRS are both involved in the conformational transition linked to the binding of p-aminobenzamidine to the thrombin active site; (2) the extent of thrombin's capacity to undergo conformational transitions in alpha-, zeta- and gamma T forms is positively correlated to the free energy of activation for hydrolysis of macromolecular substrates interacting with both the catalytic domain and the FRS.

Amino Acid Sequence↗