Amidase activity and thermal stability of human thrombin

Previous studies of amidase activity of human alpha-thrombin have yielded variable results and the decrease of this activity as a function of time and temperature has never been quantified. As this protease is an efficient tool in biochemistry and biotechnology thanks to its extreme selectivity, amidase activity and stability of thrombin were investigated with the synthetic substrate Tos-Gly-Pro-Arg-pNa. Enzyme activity as a function of temperature showed an optimum peak at 45 degrees C. The pH dependence of the activity showed a maximum around 9.5. The addition of NaCl promoted an increase of the activity. Stability of thrombin decreased rapidly when increasing the temperature from 25-45 degrees C and when diluting the enzyme. The presence of glycerol and ethylene glycol promoted a small increase of thrombin half life, whereas polyethylene glycol had a more pronounced positive effect even at very low concentrations.     

Thrombin regulation in children differs from adults in the absence and presence of heparin

The physiologic mechanisms that protect children from thromboembolic complications are not known. We investigated the regulation of thrombin in children because of its central importance to thrombosis. The capacity to generate thrombin in vitro (chromogenic assay) was decreased by 26% in plasmas from children (1-16 yrs; n = 102) compared to adults ([20-45 yrs; n = 20; p < 0.001]). The addition of purified prothrombin to plasmas from children increased thrombin generation to adult values. The capacity of plasmas to inhibit 125I-alpha-thrombin was increased by 21% in children compared to adults (p = 0.020), with significantly more thrombin complexed to alpha 2-macroglobulin (alpha 2M) in children. When DVT occur in children, adult guidelines for heparin therapy are used. At low heparin concentrations (0.1 and 0.2 U/ml), thrombin generation was decreased by 30% in children compared to adults (p < 0.001). At high heparin levels (0.4 U/ml), thrombin generation was negligible in all plasmas. ATIII inhibited over 95% of thrombin in all plasmas in the presence of heparin. In summary, thrombin regulation differs in children from adults and may protect children from thromboembolic complications. When DVT do occur, heparin requirements may differ in children compared to adults.     

Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis"

The murine/human chimeric monoclonal antibody fragment (c7E3 Fab) blocks GPIIb/IIIa and alpha v beta 3 receptors, inhibits platelet aggregation, and decreases the frequency of ischemic events after coronary artery angioplasty in patients at high risk of suffering such events. Although inhibition of platelet aggregation is likely to be the major mechanism of c7E3 Fab's effects, since activated platelets facilitate thrombin generation, it is possible that c7E3 Fab also decreases thrombin generation. To test this hypothesis, the effects of c7E3 Fab and other antiplatelet agents were tested in a thrombin generation assay triggered by tissue factor. c7E3 Fab produced dose-dependent inhibition of thrombin generation, reaching a plateau of 45-50% inhibition at concentrations > or = 15 micrograms/ml. It also inhibited thrombin-antithrombin complex formation, prothrombin fragment F1-2 generation, platelet-derived growth factor and platelet factor 4 release, incorporation of thrombin into clots, and microparticle formation. Antibody 6D1, which blocks platelet GPIb binding of von Willebrand factor, had no effect on thrombin generation, whereas antibody 10E5, which blocks GPIIb/IIIa but not alpha v beta 3 receptors decreased thrombin generation by approximately 25%. Combining antibody LM609, which blocks alpha v beta 3 receptors, with 10E5 increased the inhibition of thrombin generation to approximately 32-41%. The platelets from three patients with Glanzmann thrombasthenia, who lacked GPIIb/IIIa receptors but had normal or increased alpha v beta 3 receptors, supported approximately 21% less thrombin generation than normal platelets. We conclude that thrombin generation initiated by tissue factor in the presence of platelets is significantly inhibited by c7E3 Fab, most likely in part through both GPIIb/IIIa and alpha v beta 3 blockade, and that this effect may contribute to its antithrombotic properties.     

Human factor Va1 and factor Va2: properties in the procoagulant and anticoagulant pathways

Human plasma factor V is heterogeneous and yields two forms of activated factor V that bind with low (factor Va1) and high affinity (factor Va2) to phospholipids. The properties of factor Va1 and factor Va2 in the anticoagulant and procoagulant pathways were evaluated by comparing their sensitivity for inactivation by APC and their ability to act as cofactor in prothrombin activation. At low phospholipid concentrations and on membranes containing low amounts of phosphatidylserine (PS), factor Va1 was inactivated by APC at 15-fold lower rates than factor Va2, both in the absence and in the presence of protein S. At high phospholipid concentrations and on membranes with more than 15 mol % PS, factor Va1 and factor Va2 were inactivated with equal efficiency. Differences between cofactor activities of factor Va1 and factor Va2 in prothrombin activation were only observed on membranes with less than 7.5 mol % PS. Due to the different phospholipid requirements of APC-catalyzed factor Va inactivation and of expression of factor Va cofactor activity in prothrombin activation, the thrombin-forming capacity of factor V1 was 7-fold higher than that of factor V2 in a reaction system containing factor Xa, prothrombin, APC, protein S, vesicles with a phospholipid composition resembling that of activated platelets, and traces of thrombin to initiate prothrombin activation. This shows that in the process of generation, expression, and down-regulation of factor Va cofactor activity on physiological membranes, the overall procoagulant activity of factor V1 can considerably exceed that of factor V2.     

The measurement of thrombin in clotting blood by radioimmunoassay

We have developed a radioimmunoassay for human thrombin using rabbit anti-human thrombin IgG. The assay can measure 2 ng thrombin/ml plasma, 500-fold more sensitive than clotting assays. Human prothrombin is less reactive in the assay than thrombin by at least four orders of magnitude, and there is no demonstrable cross-reactivity with human factor Xa, the clotting factor structurally most similar to thrombin. The assay does not detect thrombin bound to anthithrombin III. Using the assay, we have demonstrated that plasma from 20 normal subjects does not contain detectable thrombin. We measured thrombin generation in clotting blood in polypropylene tubes and observed that thrombin appears (approximately equal to 3 ng/ml) within 45 S-5 min after venipuncture. This material is thrombin, not intermediates of prothrombin activation, since it disappears after addition of heparin, which promotes thrombin antithrombin III complex formation. After a plateau of 2-10 min, there is further thrombin generation, which results in clotting after 15-27 min at a level of 40-50 ng thrombin/ml. The thrombin generated 9-25 min before clotting may activate factors V and VIII and stimulate platelet aggregation and release. In contrast, the cascade hypothesis assigns a role for thrombin only late in blood clotting. Radioimmunoassay of thrombin and other clotting factors will be useful for clinical and physiological studies of blood clotting especially since the assay seems specific for thrombin and is independent of other activities that affect bioassays.     

Clinical studies and thrombin generation in patients homozygous or heterozygous for the G20210A mutation in the prothrombin gene

A genetic variation in the prothrombin gene, the G-->A transition at nucleotide 20210, is a risk factor for venous thrombosis in heterozygotes and is associated with increased prothrombin activity. The homozygous phenotype and the extent of thrombin generation in heterozygous and homozygous subjects are unknown. We investigated a family that included 2 homozygous and 5 heterozygous carriers of the 20210 A allele. The homozygous propositus and his presumably heterozygous father suffered from deep-vein thrombosis. His presumably heterozygous mother and his homozygous sister had recurrent phlebitis at a young age. The remaining 5 affected family members are still asymptomatic. We studied thrombin generation in the family and in 22 unrelated carriers of the 20210 A allele by measuring (1) prothrombin fragment F1+2 (F1+2) as an index of ongoing thrombin generation and (2) the endogenous thrombin potential (ETP) as an index of the possible thrombin-forming capacity. Their F1+2 levels were not different from those of age-matched controls, and thus, ongoing hemostatic system activation was not detectable. A significantly increased ETP was found in the heterozygous carriers of the 20210A allele compared with the controls (527.8+/-114.9 versus 387+/-50.1 nmol/L x min, P<0.0001). In the 2 homozygotes, the ETP was almost twice (639 and 751 nmol/L x min, respectively) as high as in the controls. We conclude that homozygosity for the G20210A mutation in the prothrombin gene is associated with a severe, albeit more benign, thrombotic diathesis compared with homozygosity for deficiencies of antithrombin, protein C, or protein S. In carriers of the 20210 A allele, the pathomechanisms leading to thrombosis should be sought in the higher amounts of thrombin that may be formed once thrombin generation is triggered, rather than in ongoing thrombin generation in vivo.     

Interaction of manganese with bovine prothrombin and its thrombin-mediated cleavage products

The binding of the paramagnetic metal, Mn(II), to bovine prothrombin and the thrombin-mediated cleavage products of prothrombin, i.e. fragment 1 and the prethrombin 1 has been investigated. Analysis of the Scatchard plots of the binding data reveals that prothrombin has two high affinity Mn(II) binding sites with a Kd of 1.2 +/- 1.0 X 10(-5) M and approximately two to three lower affinity Mn(II) sites with a Kd of 1.3 +/- 1.0 X 10(-4) M. Positive cooperativity in Mn(II) binding to prothrombin was observed for the strong sites. Fragment 1, the phospholipid-binding region of prothrombin, possesses two high affinity Mn(II) sites with a Kd of 2.2 +/- 1.0 X 10(-5) M and at least two lower affinity sites with a Kd of approximately 2.5 +/- 1.0 X 10(-4) M. Positive cooperativity was not observed for the binding of Mn(II) to fragment 1. Prethrombin 1 binds one Mn(II) with a Kd of 3.2 +/- 1.0 X 10(-4) M. Using the values of free Mn(II) concentration, as determined by EPR measurements and the observed enhancements of the water proton relaxation rates at various concentrations of Mn(II) and protein, the binary enhancement values (epsilon b) of the metal-protein complexes were obtained. The extrapolated values are 11 +/- 0.4 for the initial prothrombin-binding sites, and 10 +/- 0.3 for the tight binding sites of fragment 1. The unique epsilon b value obtained for prethrombin 1 was 5.3 +/- 0.7. When Mn(II) was used in a Factor Xa-metal ion-phospholipid system for activation of prothrombin, the rate of generation of thrombin was less than or equal to 5% of that obtained when Ca(II) was employed in this activation system. Addition of Mn(II) to the same activation system containing Ca(II) resulted in a marked decrease in the rate of thrombin generation, suggesting that Mn(II) probably competes for the same sites on prothrombin as Ca(II). In agreement with this is the observation that the Mn(II) sites on prothrombin could be displaced by Ca(II) at high concentrations of Ca(II).     

Interaction of short chain and long chain fatty acid phosphoglycerides and bile salts with prothrombin

An equimolar mixture of phosphatidylserine and (dioleoyl) phosphatidyl-ethanolamine could substitute for brain cephalin preparations in the single stage prothrombin assay. However, no clot promoting activity was observed on the addition of any of the individual long chain fatty acid-containing phospholipids. Short chain fatty acid-containing phospholipids, such as diheptanoylphosphatidylcholine, diheptanoylphosphatidylethanolamine, diheptanoylphosphatidic acid, and dihexanoylphosphatidylcholine, or dihexanoylphosphatidylethanolamine were inhibitory under all conditions studied. Similar effects of these two general classes of phospholipids were observed in a two-stage thrombin generation system, in which a mixture of bovine Factor Xa, Factor Va, and Ca2+ were interacted with prothrombin. In the presence of 25 mM Ca2+, dioleoylphosphatidic acid or brain phosphatidylserine alone, and with other long chain phospholipids, formed complexes with bovine plasma prothrombin. On the other hand, dioleoyl-, diheptanoyl- or dihexanoylphosphatidylcholine under comparable conditions showed no binding to prothrombin. There appeared to be a small degree of binding of diheptanoylphosphatidic acid to prothrombin, but it was insufficient to cause any significant change in apparent molecular weight of prothrombin. A mixture of prothrombin, Factor V, diheptanoylphosphatidic acid/diheptanoylphosphatidylcholine and Ca2+ eluted in the void volume of Sephadex G-200, but showed a much reduced coagulant activity. Though a net negative charge on the phospholipid surface is required for phospholipid-protein interactions, this does not necessarily promote coagulant activity. Bile acids and bile salts, such as cholic acid, deoxycholic acid, taurocholic acid, glycocholic acid, lithocholic acid and dehydrocholic acid, exerted varying levels of stimulation on the prothrombin assay and thrombin generation system, but were not as effective as the phospholipids. Interestingly, no interaction of these bile acids or salts with prothrombin was noted in the presence of Ca2+. The results of these experiments suggest that negatively charged micelles per se are not sufficient for binding alone and that other chemical and physical characteristics of phospholipids are of prime importance.     

Thrombin generation measured ex vivo following microvascular injury is increased in SLE patients with antiphospholipid-protein antibodies

Antiphospholipid-protein antibodies (APA) include lupus-type anticoagulant (LA) and antibodies recognizing complexes of anionic phospholipids (e.g. cardiolipin) and proteins (e.g. prothrombin and beta2-glycoprotein I). The presence of APA is associated with an increased risk of both arterial and venous thrombosis. However, the pathogenic mechanism leading to thrombosis in patients with APA remains unclear. We studied 32 patients with systemic lupus erythematosus (SLE) who were divided into two groups depending on the presence (n = 19) or absence (n = 13) of APA. Healthy volunteers (n = 12) matched by age and sex served as controls. In all subjects LA and IgG class anticardiolipin antibodies (ACA) were determined. Thrombin generation was monitored ex vivo measuring fibrinopeptide A (FPA) and prothrombin fragment F1 + 2 (F1 + 2) in blood emerging from a skin microvasculature injury, collected at 30 second intervals. In subjects with antiphospholipid antibodies mean FPA and F1 + 2 concentrations were significantly higher at most blood sampling times than in controls. In some SLE patients with APA the process of thrombin generation was clearly disturbed and very high concentrations of fibrinopeptide A were detected already in the first samples collected. Two minutes after skin incision SLE patients without APA produced slightly more FPA, but not F1 + 2, as compared to healthy subjects. Mathematical model applied to analyze the thrombin generation kinetics revealed that APA patients generated significantly greater amounts of thrombin than healthy controls (p = 0.02 for either marker). In contrast, in the same patients generation of thrombin in recalcified plasma in vitro was delayed pointing to the role of endothelium in the phenomenon studied. In summary, these data show for the first time that in SLE patients with antiphospholipid-protein antibodies thrombin generation after small blood vessel injury is markedly increased. Enhanced thrombin generation might explain thrombotic tendency observed in these patients.     

Reproducibility and simplification of 13C-octanoic acid breath test for gastric emptying of solids

Factor V (FV) activation is the result of cleavages at Arg709, Arg1018 and Arg1545 by thrombin or FXa. The relative importance of these cleavages in tissue factor (TF) induced thrombin generation in plasma and in a purified system was elucidated with recombinant FV in which the three sites had been eliminated one by one or in combinations. The mutants were analyzed with a clotting assay using FV-deficient plasma and in a TF induced thrombin generation system using plasma or purified components. Surprisingly, in the standard FV clotting assay, all mutants gave similar clotting activities and the thrombin generation curves obtained with wild-type and thrombin-resistant FV were similar. Differences in clotting activities and thrombin generation patterns between wild-type and thrombin-resistant FV were only observed when lower TF concentrations were used. The thrombin generation curve obtained in plasma containing wt FV was characterized by a short lag phase and a subsequent phase of rapid thrombin generation (propagation phase). The Arg709 to Gln mutation yielded a slightly prolonged lag phase and the rate of thrombin generation during the propagation phase was approximately 5-fold lower than that observed with wt FV. The Arg1018 to Ile mutation only slightly affected the thrombin generation curve, whereas the Arg1545 to Gln mutation yielded a prolonged lag phase and decreased maximum thrombin activity. Thrombin-resistant FV (mutated at all three sites) yielded a prolonged lag phase and poor thrombin generation during the propagation phase. The purified system further demonstrated the importance of the three cleavage sites for rapid and sustained thrombin generation. The results demonstrate that cleavages at positions 709, 1018 and 1545 are not required for assembly of a FXa-FV complex expressing low but significant prothrombinase activity but that all three sites in different ways are important for the creation of a FVa which maximally supports the FXa-mediated activation of prothrombin.     

Drinking in Atlantic salmon presmolts and smolts in response to growth hormone and salinity

Both thrombin and the synthetic tetracapeptide thrombin receptor-activating peptide (TRAP), recently described as a peptide mimicking the new amino terminus created by cleavage of the thrombin receptor, stimulated the proliferation of human umbilical vein endothelial cells (HUVEC) in culture. Although to a lesser extent, F-14, a tetradecapeptide representing the residues 365-378 of human prothrombin, also promoted HUVEC growth, thereby demonstrating that thrombin can stimulate HUVEC growth via both a proteolytic and a nonenzymatic pathway. Thrombin-TRAP, and F-14-induced HUVEC growth were inhibited by a thrombin receptor oligodeoxynucleotide antisense, showing that the growth-inducing effects of all 3 compounds were mediated through the same thrombin receptor. Thrombin and TRAP also stimulated intracellular Ca2+ increase, monolayer permeability increase, and prostacyclin release in HUVEC. None of these effects was observed with F-14 suggesting that thrombin-induced intracellular Ca2+ release, permeability increase, and prostacyclin release in HUVEC required catalytic cleavage of the receptor, whereas thrombin-induced growth might also be due to activation of the thrombin receptor through a nonproteolytic pathway.     

Improvement of beef tenderness and quality traits with calcium chloride injection in beef loins 48 hours postmortem

Boneless strip loin subprimals (n = 24) were fabricated from 12 USDA Standard yield grade 2 carcasses at a commercial beef processing facility and processed 48 h postmortem to determine the effect of injection of 200 or 250 mM calcium chloride (CaCl2) solution at 5% (wt/wt) on beef quality traits. One-third of the subprimal served as the control; the remaining portion was injected with either 200 or 250 mM CaCl2 at 5% (wt/wt). The CaCl2 concentration treatment was randomly assigned to strip loins fabricated from either the right or left side of the carcass. After 7 or 14 d of postmortem storage at 2 degrees C, 2.5-cm-thick steaks were cut from each control and treated portion of the subprimals and evaluated for Warner-Bratzler shear (WBS) force, retail display characteristics, Minolta colorimeter L*, a*, and b* values, and trained sensory panel ratings. Treatment of the muscle with 250 mM CaCl2 increased (P < .05) trained sensory panel tenderness and beef flavor scores, and both CaCl2 concentrations decreased WBS force values, when compared with the control. Scores for color, uniformity, and browning in the retail display case did not differ (P > .05) for the 200-mM treatment compared with the control. Scores for discoloration in the retail display decreased (P < .05) for all three-way interactions of CaCl2 concentration, aging time, and display time after d 2 (except 7-d control, which remained the same [P > .05] d 1 through 5). The L* values did not differ (P < .05) for interactions of CaCl2 concentration, x aging time, and retail display.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of thrombin generation in plasma by inhibitors of factor Xa

A series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations. The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.     

Comparative effects of enoxaparin and unfractionated heparin in healthy volunteers on prothrombin consumption in whole blood during coagulation, and release of tissue factor pathway inhibitor

In a randomized crossover study twelve healthy male volunteers (23.5 +/- of 4.8 years, 73.0 +/- 6.4 kg, 180.8 +/- 5.7 cm) received one subcutaneous injection of either enoxaparin (EN) at 40 mg or 1 mg kg-1, or unfractionated heparin (UH) at 5,000 IU at one week intervals. Area under curves (AUC) of Anti-Xa and Anti-IIa activities correlated with EN dose. The relative effectiveness of EN versus UH 5,000 U as assessed by AUC ratio (EN/UH) was 7 and 15 for Anti-Xa activity, 1.3 and 3.1 for Anti-IIa activity after sc injection of EN 40 mg (4,000 Anti-Xa IU and 1,200 Anti-IIa U) and 1 mg kg-1 (7,300 +/- 640 Anti-Xa IU and 2,190 +/- 290 Anti-IIa IU) respectively. In volunteers receiving EN, a dose dependent inhibition of thrombin generation rate in platelet depleted plasma (PDP), measured with a new and simple chromogenic thrombin generation assay, was observed when compared with baseline values. Similarly, intrinsic prothrombin activation in whole blood, evidenced by measuring residual factor II in serum 2 hours after clotting (prothrombin consumption test: PC), was inhibited in a dose dependent manner. In UH treated volunteers, although the inhibition of thrombin generation rate in PDP was similar to that observed with EN 40 mg, prothrombin consumption in whole blood was not significantly modified. Tissue factor pathway inhibitor (TFPI) activity release was increased similarly for UH and EN 40 (1.4 fold increase above baseline values) and 1.9 fold for the higher dose of EN. The discrepancy between prothrombin consumption in whole blood and inhibition of thrombin generation rate in PDP in the UH and not in the EN group strongly suggests that UH and not EN is influenced by the presence of a platelet component. This could be formed during thrombin induced platelet activation. Platelet factor 4 is a possible candidate. Another hypothesis involves the role of TFPI-UH complex anticoagulant activity which might be inhibited more during whole blood coagulation than the TFPI-EN complex.     

Binding of quinacrine to acidic phospholipids and pancreatic phospholipase A2. Effects on the catalytic activity of the enzyme

Binding of quinacrine to phospholipids and porcine pancreatic phospholipase A2 (PLA2) was investigated using fluorescence resonance energy transfer, Langmuir films, assay for the enzymatic activity, and molecular modeling. No significant binding of this drug to the zwitterionic phosphatidylcholine was observed whereas a high affinity for acidic phospholipids was revealed by quenching of pyrene-labeled phospholipid analogues. Partial reversal of this binding was observed due to the addition of 4 mM CaCl2. Quinacrine efficiently and independently of the lipid surface pressure penetrated into monolayers of phosphatidylglycerol while only a weak penetration into phosphatidylcholine films was evident. Quinacrine also bound to eosin-labeled PLA2, and the addition of 4 mM CaCl2 reversed this interaction almost completely. In the presence of acidic phospholipids both the drug and the enzyme were attached to the lipid surface. Studies on the influence of quinacrine on the activity of PLA2 toward pyrene-labeled phospholipid analogues revealed that the hydrolysis of phosphatidylcholine was progressively reduced as a function of increasing [quinacrine]. At low [CaCl2] and low quinacrine:lipid molar ratios (<1:5) quinacrine enhanced slightly the rate of hydrolysis of acidic phospholipids whereas at higher drug:lipid molar ratios (>1:2) an inhibition was observed. In the presence of 1 mM CaCl2 quinacrine inhibited PLA2-catalyzed hydrolysis of phosphatidylglycerol only when the drug:lipid molar ratio exceeded 1:1. The presence of 4 mM CaCl2 abolished nearly completely the inhibition with all the substrate analogues used. Our data suggest that the inhibition of PLA2 by quinacrine is due to its binding to the enzyme. This is supported also by molecular modeling which suggested a binding site for quinacrine close to the active site and Ca2+ binding site of the enzyme. Importantly, our data indicate that quinacrine binds avidly to acidic phospholipids and their presence may influence the drug-enzyme interaction and the inhibition of the enzyme action. Accordingly, presence of quinacrine may interfere also with other processes that require the presence of acidic lipids and/or Ca2+, such as the function of the nicotinic acetylcholine receptor.     

Endonucleolysis of chromatin in rat thymus and spleen lymphocytes after irradiation

The level of soluble DNA fragments (polydesoxynucleotides) in thymus and spleen lymphocytes is enhanced 12 h after the whole-body X-ray irradiation in dose of 0.5 and 1 Gy. The level of Ca2+, Mg(2+)-DNAase activity in lysed cells extracts, measured with calf spleen DNA, was enhanced in the region of 0.1-0.5 mM CaCl2 in incubation medium and decreased at 5 mM CaCl2. Monitoring of DNA fragmentation by electrophoresis in agarose gels showed the presence of fragments with approximately 200, 400, 800 bp length. X-ray induced DNA fragmentation is supposed to be associated with Ca2+, Mg(2+)-endonuclease activation.     

Thrombin receptor activating peptide does not stimulate platelet procoagulant activity

Platelets after challenge with alpha-thrombin alone, collagen alone or thrombin/collagen mixture were observed to increase the rate of activation of prothrombin by factor Xa in the presence of factor Va and calcium ion (platelet procoagulant activity) by a maximum of 25, 45 and 110 fold, respectively. The increase in platelet procoagulant activity due to these agonists has been described previously and arises from increased expression of phosphatidylserine on the platelet surface. When platelets were treated with the thrombin receptor activating peptide (TRAP) (SFLLRNPNDKYEPK), alone or in the presence of collagen or thrombin, no change in platelet procoagulant activity was observed at concentrations of TRAP sufficient to cause increased intracellular calcium levels and protein phosphorylation in a manner similar to that of thrombin. In addition, no increase in platelet procoagulant activity was seen upon treatment with TRAP in the presence of inactivated thrombin (PPACK-thrombin). These results suggest that the thrombin-mediated increase in procoagulant activity may be due to activation of a thrombin receptor distinct from the recently cloned G-protein-coupled receptor, or to other proteolytic events on the platelet surface.     

Role of gamma-carboxyglutamic acid. An unusual protein transition required for the calcium-dependent binding of prothrombin to phospholipid

A first order calcium-dependent transition can be monitored by a decrease in the intrinsic fluorescence of the isolated "pro" (Fragment1) region of prothrombin. The maximum fluorescence change is -40% for Fragment 1, and only about -6% for whole prothrombin. The most remarkable features of this transition are its rate and activation energy. The half-life for the transition at 0 degrees is about 100 min, and the temperature dependence shows an activation energy of 21 kcal/mol. The rate constant for the forward reaction is zero order in calcium and is not affected by the presence of phospholipid membranes. The equilibrium for the transition, however, is affected by phospholipid. At 30 degrees, [Ca]eq (the calcium concentration where half of the protein has undergone the transition) is 0.4 mM and the Hill coefficient is 2.6. Under the same conditions but in the presence of phospholipid [Ca]eq is 0.24 mM and the Hill coefficient is about 4.5. The transition is triggered by binding 3 or 4 calcium ions. The rate of Fragment 1 binding to phospholipid vesicles was tested using gel filtration techniques at 0 degrees. The rate constants, activation energy, and [Ca]eq values for this process were shown to correspond to the properties of the fluorescence change. The rate constants, activation energy, and Hill coefficients for binding of whole prothrombin to phospholipid correspond to the same parameters for Fragment 1 but the [Ca]eq values are lower. At 0 degrees, the [Ca]eq is 0.19 mM for the prothrombin transition and 0.1 mM for the transition in the presence of phospholipid. These results demonstrate that Fragment 1 and prothrombin undergo a transition when exposed to calcium ions which necessarily precedes protein-phospholipid interactions. In addition to its role in determining the correct protein structure, calcium plays a second role in prothrombin-phos-pholipid interaction which is in the actual formation of the protein-phospholipid bond. The [Ca]eq for binding protein (after its transition) to phospholipid is about 0.06 mM.     

The generation of fibrinopeptide A in clinical blood samples: evidence for thrombin activity

Plasma fibrinopeptide A (FPA) concentrations were measured in clinical blood samples incubated in the collecting syringe for different time periods before addition to heparin and Trasylol, and the rate of in vitro generation of FPA was calculated as the mean increment in FPA concentration per minute over the linear portion of the generation curve. 36 normal individuals had a mean plasma FPA level of 0.64 +/- 0.56 pmol/ml and an FPA generation rate of less than 0.5 pmol/ml per min. Clinical samples with elevated plasma FPA levels manifested slow (less than 1 pmol/ml per min) (28 patients) or rapid FPA generation (greater than 1 pmol/ml per min) (33 patients). Slow FPA generation was found in 10/10 patients with venous thrombosis, in 4/4 with aortic aneurysm, and in several patients with acquired hypofibrinogenemia. In one such patient, addition of fibrinogen resulted in rapid FPA generation whereas thrombin addition was without effect. Rapid FPA generation was generally linear, was usually associated with slower fibrinopeptide B generation and was inhibited by parenteral or in vitro heparin. It is thought to reflect increased thrombin activity and was seen in patients with pulmonary embolism, active systemic lupus erythematosus, renal transplant rejection, and after infusion of prothrombin concentrates. The initial rate of FPA cleavage by thrombin at fibrinogen concentrations from 0.05 to 4 mg/ml showed little change between 2 and 4 mg/ml with a Km of 2.99 muM. At a fibrinogen concentration of 2.5 mg/ml the FPA cleavage rate was 49.2 +/- 1.6 nmol/ml per min per U of thrombin. Exogenous thrombin added to normal blood generated 21.7 nmol/ml per U of thrombin FPA in the first minute with a nonlinear pattern reflecting inactivation of thrombin and the presence of alternative substrates. Hence, the thrombin concentration in the blood cannot be calculated from the FPA generation rate. The FPA generation rates in clinical samples with rapid generation (1-28 pmol/ml per min) could be produced by 2 X 10(-5) to 5.6 X 10(-4) thrombin U/ml acting on purified fibrinogen at physiological conditions of pH, ionic strength, and temperature.     

The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venon and characterization of the new active intermediates

Bovine prothrombin was activated, in both the absence and presence of dissopropyphosphofluoridate (DEP) and benzamidine, by an activator which was highly purified from the venom of Echis carinatus (saw-scaled viper, ECV). The process of activation was monitored by sodium dodecysulfate (SDS)-polyacrylamide gel electrophoresis, and the reaction products were isolated and chemically characterized. In the absence of the inhibitors, prothrombin yielded two fragments with molecular weights of 28,000 and 57,000, of which the former was the N-terminal fragment of the zymogen and the latter was intermediate 1, consisting of a single polypeptide chain. Intermediate 1 was subsequently converted to an active intermediate, named intermediate ECV, without decrease of molecular weight. This new intermediate ECV, which showed little clotting activity but a strong alpha-N-tosyl-L-arginine methyl ester (TAME)-esterolytic activity and which bound with hirudin or antithrombin III, consisted of two polypeptide chains with molecular weights of 35,000 of 27,000 daltons. The former was indentified as the thrombin B chain with the N-terminal sequence Ile-Val-Glu-Gly and C-terminal serine, and the latter was a fragment with N-terminal Ser-Gly-Gly, linked to the thrombin A chain. On prolonged incubation, intermediate ECV autocaralytically yielded a fragment (inner fragment) of 14,000 daltons with N-terminal serine and the clotting enzyme alpha-thrombin [EC 3.4.21.5], which consists of A and B chains. In the presence of the inhibitors, intermediate ECV and the N-terminal fragment were accumulated in the activation mixture. On the other hand, when prothrombin was activated by the venom activator in the presence of hirudin, antithrombin III, or p-nitrophenyl p'-guanidinobenzoate, it did not yield any fragments but was converted to a derivative with two polypeptide chains having molecular weights of 51,000 and 34,000 daltons, of which the former consisted of N-terminal fragment, the inner fragment, and thrombin A chain, and the latter was thrombin B chain. This new prothrombin derivative, named prothrombin ECV, formed a high-molecular-weight complex, associating with antithrombin III. The complex was not dissociable even in the presence of SDS. Moreover, prothrombin ECV reacted with p-nitrophenyl p'-guanidinobenzoate. On the basis of the results described above, the mechanism of activaton of prothrombin by Echis carinatus venom activator can be summarized as follows: The venom activator first cleaves an Arg-Ile bond liniking thrombin A and B chains in the zymogen molecule, forming an active derivative, prothrombin ECV. This active derivative converts autocatalytically to intermediate ECV, liberating the N-terminal fragment, and active intermediate ECV generates alpha-thrombin, releasing the inner fragment. Thus, only a single peptide bond cleavage along the polypeptide chain of prothrombin is associated with activation by the venom activator...