Validation of a telephone questionnaire for Parkinson''s disease

Unfractionated as well as low-molecular-weight heparins (LMWH) are known to cause an increase in blood levels of tissue factor pathway inhibitor (TFPI). To study the effect of a newly developed supersulfated LMWH (IK-SSH, Iketon Farmaceutici) on TFPI concentrations in human plasma, the compound was injected into volunteers at doses of 0.14, 0.33 and 0.66 mg/kg intravenously or 0.33, 0.66 and 1.0 mg/kg subcutaneously. At certain known times blood was drawn and plasma levels of both total and free TFPI were measured using enzyme-linked immunosorbent assay methodology. Baseline plasma concentrations of TFPI were 72.2+/-3.1 ng/ml for total and 10.8+/-0.8 ng/ml for free TFPI. Intravenous or subcutaneous injection of IK-SSH led to a strong and long-lasting rise in TFPI levels which were increased more than 5-fold for total TFPI and more than 30-fold for free TFPI. Maximum TFPI levels were reached 5-10 min after intravenous and 60 min after subcutaneous administration. IK-SSH caused prolongation of ex-vivo clotting times in the APTT and Heptest assay, whereas thrombin time was not affected. Anticoagulant actions of IK-SSH showed a significant correlation to plasma concentrations of TFPI and they are thought to be based at least partially on the release of TFPI from vascular sites.     

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.     

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.     

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.     

Persistent thrombin generation in humans during specific thrombin inhibition with hirudin

BACKGROUND: The degree to which antithrombotic drugs suppress thrombin generation is unknown. Because hirudin, unlike antithrombin III, binds intravascular thrombin rapidly and selectively to yield a circulating inactive complex of 3- to 4-hour half-life, we used intravenous hirudin in humans to investigate the course of thrombin generation during and early after anticoagulation with this potent, direct antithrombin. METHODS AND RESULTS: Intravascular thrombin was measured with an ELISA for the thrombin-hirudin complex formed during and for 18 hours after stopping a 6-hour infusion of hirudin at 0.1, 0.2, and 0.3 mg.kg-1.h-1 in three groups of six patients each. With free hirudin in 20- to 10,000-fold molar excess of thrombin and peak activated partial thromboplastin times of 2.3 to 3.0 times baseline, mean plasma thrombin-hirudin complex increased from 794 +/- 85 pg/mL (mean +/- SEM) 15 minutes after the start of the infusion to 1617 +/- 151 pg/mL at 6 hours of infusion to 2667 +/- 654 pg/mL at 24 hours. During the 24-hour observation period, plasma concentration of fragment 1.2 (the peptide released during conversion of prothrombin to thrombin) never fell below baseline but rather increased transiently during the hirudin infusion. Plasma concentrations of thrombin-antithrombin III complex (in ng/mL) decreased from 4.34 +/- 0.40 at baseline to 1.64 +/- 0.13 at 6 hours (P < .001) and gradually increased after stopping the infusion to 5.7 +/- 0.87 at 24 hours (nonsignificant compared with baseline). CONCLUSIONS: Measurement of thrombin-hirudin complex may be used as a marker of thrombin generation in humans. Persistent accumulation of thrombin-hirudin complex and generation of fragment 1.2 during and after completion of potent anticoagulation with hirudin suggest thrombin generation is not blocked by high-affinity thrombin inhibition. The persistent formation of thrombin during declining plasma levels of hirudin may contribute to the pathogenesis of rethrombosis early after antithrombin therapy or during inadequate anticoagulation.     

The procoagulant activity of red blood cells from patients with severe preeclampsia

OBJECTIVE: Our purpose was to determine whether red blood cells from patients with severe preeclampsia may exhibit increased membrane exposure of procoagulant phospholipids (i.e., phosphatidylserine), which may initiate intravascular clotting and platelet activation. STUDY DESIGN: The study group comprised 28 women: 9 with severe preeclampsia in the third trimester of pregnancy, 10 normotensive with uncomplicated pregnancies, and 9 age-matched, nonpregnant, healthy women. The exposure of phosphatidylserine on the outer membrane phospholipid layer was analyzed with use of isolated, washed red blood cells that were added as a source of phospholipids to a "prothrombinase" coagulation complex. RESULTS: The resultant thrombin formed was measured by an amidolytic assay. Thrombin generation significantly increased on the addition of red blood cells from women with preeclampsia (741 +/- 132 mU/ml/min) compared with red blood cells from normotensive pregnant (422 +/- 228 mU/ml/min) and nonpregnant women (316 +/- 268 mU/ml/min, p = 0.0008). CONCLUSION: This study indicates that in patients with preeclampsia the red blood cells exhibit a significant procoagulant surface that may trigger thrombin formation, thereby playing a role in the hypercoagulable state.     

Effects of cardiopulmonary bypass and deep hypothermic circulatory arrest on the thyroid axis during and after repair of congenital heart defects: preservation by deep hypothermia?

YM-60828 was found to potently inhibit human factor Xa following oral administration. YM-60828 showed high affinity for factor Xa (Ki = 1.3 nM), but did not affect thrombin (Ki > 100 microM). YM-60828 doubled factor Xa clotting time, prothrombin time (PT) and activated partial thromboplastin time (APTT) at 0.10, 0.21, 0.24 microM, respectively. Importantly, it did not prolong thrombin time at 100 microM. YM-60828 also inhibited factor Xa in the prothrombinase complex with an IC50 value of 7.7 nM. In addition to its anticoagulant activity, YM-60828 inhibited platelet aggregation induced by various agonists (IC50 = 3 to 23 microM). Squirrel monkeys were used to study the ex vivo anticoagulant activity and pharmacokinetic properties of YM-60828. One hour after oral administration at 3 mg/kg, YM-60828 strongly prolonged PT and APTT by 4.8- and 1.9-fold, respectively, and plasma concentration reached 788 +/- 167 ng/ml. Bioavailability was calculated to be 20.3%. These results strongly suggest that YM-60828 will be a valuable orally active and potent anticoagulant agent showing potential antithrombotic activity.     

Value of irradiation of neck nodes metastases. Part I. Treatment of palpable nodes

The N-methyl-D-phenylalanyl-L-prolyl-arginine-aldehyde sulfate tripeptide-aldehyde (GYKI-14766) is an anticoagulant with specific thrombin inhibitor action. The molecule proved to be effective in rabbits, rats and dogs upon i.v. administration. Chromogen-substrate assay was developed for monitoring of biologically active tripeptide-inhibitor GYKI-14766 in plasma. The assay based on the inhibition of the active center of the thrombin enzyme, so it is suitable also for the assay of all those active metabolites which inhibit thrombin by a mechanism similar to the active parent compound. The chromogen substrate assay was performed in a range of 0.625-10 micrograms/ml GYKI-14766 in dog plasma. The assay was employed in pharmacokinetic study in dogs after i.v. administration. The data obtained in the chromogen-substrate assay were analyzed according to a one-compartment model. The major parameters of the plasma level studies were: D/V = 8.6 microEqv/ml t1/2 = 30.8 min AUC = 380 min microEqv/ml.     

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.     

Activation of clotting factor XI without detectable contact activation in experimental human endotoxemia

Evidence of factor XI (FXI) activation in vivo is scarce. In addition, it remains uncertain whether thrombin, factor XIIa (FXIIa), or perhaps another protease is responsible for FXI conversion. We investigated the activation of FXI in eight healthy volunteers after infusion of a low dose of endotoxin (4 ng/kg of body weight). Activation of prekallikrein FXII, FXI, and prothrombin was measured with sensitive enzyme-linked immunosorbent assays (ELISAs), and FXI activation was measured with a novel enzyme capture assay that detects noncomplexed FXIa. Activation of FXI was apparent with a significant plasma peak level of noncomplexed FXIa of 10 to 11 pmol/L at 1 and 2 hours after endotoxin infusion, followed by a gradual increase in FXIa-FXIa inhibitor complexes, measured in the ELISAs, with a summit of 11 to 15 pmol/L at 6 and 24 hours, respectively. In accordance with previous studies, thrombin generation was detected 1 hour after endotoxin infusion to become maximal after 3 to 4 hours. In contrast, we did not find any evidence of contact activation, because markers of activation of prekallikrein and FXII remained undetectable. From the FXIa data a theoretical model was constructed which suggested that inhibition of FXIa does not take place in the plasma compartment, but is localized on a surface. These data provide the first evidence for FXI activation in low-grade endotoxemia and suggest that FXI is activated independently of FXII.     

Depletion of intravascular pools of tissue factor pathway inhibitor (TFPI) during repeated or continuous intravenous infusion of heparin in man

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of the extrinsic coagulation system. TFPI is increased several-fold in postheparin plasma and thereby thought to contribute significantly to the antithrombotic action of heparin. The present study was conducted to investigate how repeated (n = 8) and continuous (n = 6) administration of heparin affect plasma TFPI and the inhibition of tissue factor (TF)-induced coagulation ex vivo in humans. Free TFPI antigen (TFPI Ag) increased from 19.2 +/- 4.0 ng/ml to 204.7 +/- 31.7 ng/ml after intravenous injection of 5000 IU of unfractionated heparin. Five repeated injections of 5000 IU of heparin at 4 h intervals caused a progressive decrease (-45 +/- 8%, p < 0.0001 for time effect) in heparin-releasable TFPI and a progressive shortening of the clotting time as determined in a dilute prothrombin time assay (dPT) (-8.7 +/- 6.1 s, p < 0.0001). The basal concentration of TFPI Ag in plasma collected immediately before each heparin injection was decreased by 29 +/- 15% (p < 0.0001), whereas the dPT was decreased by 6.9 +/- 3.5 s (p < 0.0001). During a 24 h continuous infusion of heparin TFPI Ag decreased from 161.5 +/- 26.0 ng/ml to 35.6 +/- 4.7 ng/ml (-77.3 +/- 5.1%) (p < 0.0001). The contribution of TFPI to the inhibition of TF-induced coagulation during heparin infusion was estimated to decrease from 60 +/- 15% to 20 +/- 10% (p < 0.0001). The present data indicate partial depletion of intravascular pools of TFPI by repeated and continuous heparin administration and thereby attenuation of its contribution to the antithrombotic action of heparin.     

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.     

Monocyte procoagulant activity induced by adherence to an artificial surface is reduced by end-point immobilized heparin-coating of the surface

Heparin-coating improves the biocompatibility of blood contacting artificial surfaces. This led us to investigate the impact of heparin-coating (Carmeda AB, Stockholm) of polymetylmetacrylate on the expression of monocyte tissue factor procoagulant activity (TF-PCA) by surface adhesion. Also, the anticoagulant effect of heparin-coating in the presence or absence of adherent procoagulant monocytes was assessed. This is of particular interest, since activation of extrinsic coagulation by adherent monocyte TF-PCA may play a significant role in thrombin generation during extracorporeal circulation. Monocytes exposed to heparin-coated or non-coated polymetylmetacrylate expressed TF-PCA. The heparin coat did not affect the rate of monocyte adhesion. However, heparin-coating reduced the induction of TF-PCA of non-adherent and adherent monocytes by 17 and 33% (p <0.001 and p <0.0003), respectively. Heparin-coating in the absence of monocytes, totally inhibited the clotting of recalcified plasma (p <0.003). In contrast, in the presence of adherent monocytes expressing TF-PCA, surface-bound heparin did not inhibit clotting. However, inclusion of heparin in a plasma concentration of 8.9 IU/ml totally inhibited the activation of coagulation. It is apparent that heparin-coating of an artificial surface is an efficient means to inhibit coagulation of recalcified plasma, but much less so when procoagulant monocytes are adherent to the coated surface. The present findings are of clinical relevance, since monocytes will adhere to blood contacting surfaces of extracorporeal circuits or to implanted vascular prostheses and subsequently express TF-PCA, and this may promote thromboembolism.     

Anticoagulant effects of hirulog, a novel thrombin inhibitor, in patients with coronary artery disease

Selective thrombin inhibitors are a new class of antithrombotic drugs that, unlike heparin, can effectively inhibit clot-bound thrombin and escape neutralization by activated platelets. Hirulog is a 20 amino acid hirudin-based synthetic peptide that has shown promise in experimental models of thrombosis. Little information is available about the effects of hirulog in patients with coronary artery disease. Forty-five patients undergoing cardiac catheterization, who were taking aspirin, were randomized to receive either (1) hirulog, 0.05 mg/kg intravenous bolus followed by 0.2 mg/kg/hour intravenous infusion until the end of the catheterization; (2) hirulog, 0.15 mg/kg intravenous bolus followed by 0.6 mg/kg/hour intravenous infusion; or (3) heparin; 5,000 U intravenous bolus. Serial activated partial thromboplastin time (APTT), prothrombin time, activated clotting time and fibrinopeptide A were measured. Hirulog produced a dose-dependent prolongation of all coagulation parameters; the 0.6 mg/kg/hour dose prolonged the APTT to 218 +/- 50% of baseline after 2 minutes and 248 +/- 50% of baseline after 15 minutes. The half-life of the effect on APTT was 40 minutes. The hirulog blood level correlated well with the APTT, prothrombin time and activated clotting time (r = 0.77, 0.73, and 0.82 respectively, all p < 0.001). Both doses of hirulog potently suppressed the generation of fibrinopeptide A (p < 0.05). There were no major hemorrhagic, thrombotic or allergic complications in patients treated with hirulog or heparin. Thus, hirulog, a direct thrombin inhibitor, provides a predictable level of anticoagulation and appears to have a potent yet well-tolerated anticoagulant profile in patients with coronary artery disease.     

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.     

Coagulation and thrombomodulin in response to exercise of different type and duration

PURPOSE: The present study was conducted to evaluate the role of the duration of exercise and the impact of the exercise type for exercise-induced activation of coagulation. METHODS: Eleven male triathletes were subjected to stepwise maximal (17 min) and 1-h maximal exercise in swimming, cycling, and running. Changes of hemostatic variable sand of plasma thrombomodulin, a marker of endothelial cell activation, were monitored. RESULTS: Irrespective of the type of exercise, alterations in markers of thrombin (prothrombin fragment 1 + 2, thrombin-antithrombin III complexes) and fibrin formation (fibrinopeptide A) were more pronounced after 1-h exercise than after stepwise maximal exercise. Hemostatic parameters rose to the highest levels after running resulting in substantial fibrin formation as indicated by fibrinopeptide A increasing from 1.33 ng.mL-1 to 2.25 ng.mL (P < 0.05) after 1-h exercise testing. Significant changes of plasma thrombomodulin were detected exclusively after running with increases from 38.2 ng.mL-1 to 44.2 ng.mL-1 (1 h, P < 0.01). CONCLUSIONS: The data demonstrated that prolonged exercise is necessary for exercise-induced activation of coagulation resulting in thrombin and fibrin formation and suggested that endothelial cell activation possibly due to mechanical factors associated with running might play a role.     

Beta2-glycoprotein I is necessary to inhibit protein C activity by monoclonal anticardiolipin antibodies

OBJECTIVE: To clarify mechanisms of the thrombosis associated with anticardiolipin antibodies (aCL), we examined the effects on activated protein C (APC) of monoclonal aCL and beta2-glycoprotein I (beta2GPI), which is required for formation of the epitopes of aCL. METHODS: We developed the chromogenic assay, in which the degradation of coagulation factor Va by APC is reflected in the reduced generation of thrombin from prothrombin, using soybean trypsin inhibitor to inhibit APC. APC activities were measured in the presence and absence of 3.4 microM beta2GPI and/or 2.5 microg/ml of IgM monoclonal aCL (EY2C9 and EY1C8) established from peripheral blood lymphocytes obtained from a patient with aCL. RESULTS: Without APC, the formed thrombin activity decreased by the addition of 3.4 microM beta2GPI. When 12.8 nM APC was added, beta2GPI partially reversed the APC-induced inhibition of thrombin generation in a concentration-dependent manner. With 3.4 microM beta2GPI, the thrombin generation in monoclonal aCL (2.5 microg/ml) decreased to 77.1-80.2% by the addition of 12.8 nM APC, but the values were above that in the control IgM (72.7%). Without beta2GPI, the APC activity was unaffected by the addition of monoclonal aCL. CONCLUSION: Beta2-glycoprotein I exhibits procoagulant activity by inhibiting APC activity and anticoagulant activity by inhibiting thrombin generation. Any further inhibition of APC activity was caused by monoclonal aCL and only in the presence of beta2GPI.     

Effectiveness of heparin in preventing thrombin generation and thrombin activity in patients undergoing coronary intervention

BACKGROUND: Thrombus is important in the pathophysiology of several complications of angioplasty, including abrupt closure and restenosis. Levels of prothrombin fragment F1.2 and fibrinopeptide A reflect thrombin generation and activity. The effect of angioplasty on levels of these markers is unclear. METHODS: Patients undergoing either balloon angioplasty (n = 30) or directional atherectomy (n = 9) were treated with heparin to maintain an activated clotting time of >300 seconds. Levels of F1.2, fibrinopeptide A, and thrombin-antithrombin complex were measured in the coronary sinus and coronary artery before and after intervention. Angiograms were reviewed for lesion morphologic characteristics and dissection. RESULTS: There was no evidence for thrombin generation or increased thrombin activity after angioplasty regardless of lesion morphologic characteristics, dissection, type of intervention, or blood sampling site. In fact, coronary sinus concentrations of F1.2 decreased after intervention (median 0.31 nmol/L; 25th percentile 0.26 nmol/L, 75th percentile 0.37 nmol/L) before intervention to 0.23 nmol/L (25th percentile 0.19 nmol/L, 75th percentile 0.34 nmol/L) after intervention (P =.002). CONCLUSIONS: Angioplasty performed in the presence of adequate heparin inhibited thrombin even when there was complex lesion morphology or dissection. These data suggest that heparin provides satisfactory thrombin inhibition during routine angioplasty.     

Melphalan estimation by quantitative thin-layer chromatography. Observations on melphalan hydrolysis in vitro and pharmacokinetics in rabbits

A simple and specific quantitative high-performance thin-layer chromatographic (HPTLC) assay for melphalan in plasma is described. This assay was linear over the investigated range of 50--3,000 ng/ml, with a minimum level of detection of 20 ng/ml. Comparison with a high-pressure liquid chromatographic (HPLC) technique yielded similar estimates for melphalan concentrations in human plasma samples. The HPTLC method, unlike the HPLC technique, does not resolve monohydroxymelphalan satisfactorily. The HPTLC method was used to determine the activation energy for in vitro melphalan hydrolysis: this was 14.5 kcal/mole. The pharmacokinetics of melphalan in rabbits were also investigated. The mean t1/2 in four animals was 32.6 +/- 10.3 (S.D.) min and following IV administration to two animals the apparent volumes of distribution were 2.20 and 1.73 l/kg.     

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.