Release kinetics of intact and degraded troponin I and T after irreversible cell damage.

PURPOSE: We characterized the release kinetics of cardiac troponin I and T in relation to lactate dehydrogenase (LDH) from cardiomyocytes before and after the transition from reversible to irreversible cell damage. METHODS: Cardiomyocytes were exposed to mild metabolic inhibition (1 mmol/L sodium azide) to induce a necrotic cell death process that is characterized by a reversible (0-12 h) and irreversible phase (12-30 h). At various time intervals cells and media were collected and analyzed for LDH activity, intact cTnI and cTnT, and their degradation products. RESULTS: During the first 12 h of metabolic inhibition, cell viability was unchanged with no release of intact cTnI and cTnT nor their degradation products. Between 12 and 30 h of azide treatment, cardiomyocytes showed progressive cell death accompanied by release of intact cTnI (29 kDa), intact cTnT (39 kDa), four cTnI degradation products of 26, 20, 17 and 12 kDa, and three cTnT degradation products of 37, 27 and 14 kDa. Possibly due to degradation, there is progressive loss of cTnI and cTnT protein that is obviously undetected by the antibodies used. CONCLUSIONS: Metabolic inhibition of cardiomyocytes induces a parallel release of intact cTnI and cTnT and their degradation products, starting only after onset of irreversible cardiomyocyte damage.

Viral vectors, tools for gene transfer in the nervous system.

Viral vectors are becoming increasingly important tools to investigate the function of neural proteins and to explore the feasibility of gene therapy to treat diseases of the nervous system. This gene transfer technology is based on the use of a virus as a gene delivery vehicle. In contrast to functional analysis of gene products in transgenic mouse, viral vectors can be applied to transfer genes to somatic, post-mitotic cells of fully developed animals. To date, five viral vector systems are available for gene transfer in the nervous system. These include recombinant and defective herpes viral vectors, adenoviral vectors, adeno-associated viral vectors and lentiviral vectors. Of these vectors herpes and adenoviral vectors are the most common in use. To date, one of the main hurdles in applying these two vector systems is the focal immune response that occurs following intraparenchymal infusion. Despite this limitation, herpes and adenoviral vectors have been used successfully to modify the physiological response to injury in several rodent models of neurodegeneration. The first purpose of this review is to describe the principles of the generation of viral vectors and to discuss the advantages and disadvantages of the viral vector systems currently in use for gene transfer in the nervous system. Secondly, we give an overview of the performance of these vectors following direct infusion in the nervous system and review the results obtained with these vectors in animal models of neurodegeneration and regeneration. The results of these initial studies have provided a framework for future experiments based on gene transfer strategies with viral vectors to study normal physiology and pathology of the nervous system.

Binding of prothrombin and its fragment 1 to phospholipid membranes studied by the solvent relaxation technique.

The phospholipid headgroup mobility of small unilamellar vesicles composed of different mixtures of phosphatidyl-L-serine (PS) and phosphatidylcholine is characterized by the solvent relaxation behavior of the polarity sensitive dyes 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and 6-palmitoyl-2-[trimethylammoniumethyl]-methylamino]naphthalene chloride (Patman). If the PS content exceeds 10%, the addition of calcium leads to a substantial deceleration of the solvent relaxation of both dyes, indicating the formation of Ca(PS)2 complexes. Addition of prothrombin and its fragment 1 leads to a further decrease of the headgroup mobility, as explained by the binding of more than two PS-molecules by a single protein molecule. Prodan monitors the outermost region of the bilayer and it clearly distinguishes between the binding of prothrombin and its fragment 1. The deeper incalated Patman does not distinguish between both proteins. The validity of the solvent relaxation technique for the investigation of the membrane binding of peripheral proteins is demonstrated by the studies of prothrombin induced changes in the steady-state fluorescence anisotropies of 1,6-diphenyl-1,3, 5-hexatriene.

Production of thrombin as a probe for mixing of phospholipids in membranes on solid supports.

Phospholipid-covered solid supports have been used successfully as model membranes in studies on blood coagulation and other research fields. In order to produce such membranes, simple exposure of the support to suspensions of phospholipid vesicles was recently introduced, but questions have remained about the process of vesicle adherence to the surface and the physico-chemical properties of the resulting membranes. Using a new technique, mixing of phospholipids in such membranes was demonstrated. A rotating, hydrophilic, silicon disc was exposed in a two-step procedure to vesicles prepared from mixtures of dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidylcholine (DOPC). Factor Xa, factor Va and prothrombin were added and the transport-limited production rate of thrombin was measured. For low surface coverage with 40% DOPS/60% DOPC, a much higher conversion rate was found if, prior to addition of coagulation factors, excess DOPC vesicles were added to fill up vacant surface area. It is concluded that DOPS is spread over the entire surface and that confluent bilayers are formed. The presented technique may also be used to measure lateral diffusion constants.

Monitoring of unbound protein in vesicle suspensions with off-null ellipsometry.

In studies on the binding of proteins to small unilamellar phospholipid vesicles (SUV), the concentration of unbound protein usually remains unknown, because the vesicles cannot be separated from the bulk solution. In the present study, this limitation was overcome by addition of a supported planar phospholipid bilayer to the cuvette containing a vesicle suspension. Ellipsometric measurement of the protein adsorption velocities on this bilayer allowed determination of the concentrations of unbound protein. At high protein concentrations the adsorption is rapidly completed and the usual null-ellipsometry is too slow to obtain well-defined initial adsorption rates. Therefore, an off-null technique was developed, allowing measurement of the adsorbed protein mass at time intervals of 20 ms. Binding of prothrombin and coagulation factor Xa was measured in SUV suspensions prepared from a 20% dioleoylphosphatidylserine (DOPS) and 80% dioleoylphosphatidylcholine (DOPC) phospholipid mixture. For prothrombin, a dissociation constant Kd = 140 +/- 27 nM (mean +/- S.E.) and maximal surface concentration gamma max = (8.9 +/- 0.8) x 10(-3) mole of protein per mole of lipid, were obtained. For factor Xa, these values were Kd = 49.6 +/- 6.3 nM and gamma max = (23.0 +/- 1.4) x 10(-3) mole of protein per mole of lipid. These binding parameters are similar to those obtained earlier for planar bilayers. Apparently, the binding of factor Xa and prothrombin is not dependent on surface curvature.

C-reactive protein as a cardiovascular risk factor: more than an epiphenomenon?

BACKGROUND: Circulating levels of C-reactive protein (CRP) may constitute an independent risk factor for cardiovascular disease. How CRP as a risk factor is involved in cardiovascular disease is still unclear. METHODS AND RESULTS: By reviewing available studies, we discuss explanations for the associations between CRP and cardiovascular disease. CRP levels within the upper quartile/quintile of the normal range constitute an increased risk for cardiovascular events, both in apparently healthy persons and in persons with preexisting angina pectoris. High CRP responses after acute myocardial infarction indicate an unfavorable outcome, even after correction for other risk factors. This link between CRP and cardiovascular disease has been considered to reflect the response of the body to the inflammatory reactions in the atherosclerotic (coronary) vessels and adjacent myocardium. However, because CRP localizes in infarcted myocardium (with colocalization of activated complement), we hypothesize that CRP may directly interact with atherosclerotic vessels or ischemic myocardium by activation of the complement system, thereby promoting inflammation and thrombosis. CONCLUSIONS: CRP constitutes an independent cardiovascular risk factor. Unraveling the molecular background of this association may provide new directions for prevention of cardiovascular events.

Mutant ubiquitin expressed in Alzheimer's disease causes neuronal death.

Ubiquitin-B+1 (UBB+1) is a mutant ubiquitin that accumulates in the neurones of patients with Alzheimer's disease (AD). Here we report on the biochemical and functional differences between ubiquitin and UBB+1 and the effect of the mutant protein on neuronal cells. UBB+1 lacks the capacity to ubiquitinate, and although it is ubiquitinated itself, UBB+1 is not degraded by the ubiquitin-proteasomal system and is quite stable in neuronal cells. Overexpression of UBB+1 in neuroblastoma cells significantly induces nuclear fragmentation and cell death. Our results demonstrate that accumulation of UBB+1 in neurones is detrimental and may contribute to neuronal dysfunction in AD patients.

Myocardial enzyme depletion in infarcted human hearts: infarct size and equivalent tissue mass.

Myocardial activities of several enzymes were measured in infarcted and non-infarcted areas of heart sections obtained from eight patients who died after acute myocardial infarction. Similar data were obtained from four patients with cardiovascular disorders who died from causes other than myocardial infarction and from six patients without previously known heart disease. It was found that both non-infarcted and infarcted tissue samples contained considerably altered enzyme activities. This finding explains the low correlations between enzymatic and histological estimates of infarct size previously reported. However, when the residual myocardial activities of different enzymes were compared with each other, a close correlation was found between creatine kinase, alpha-hydroxybutyrate dehydrogenase, and aspartate aminotransferase. It appears that the pathological changes in the myocardial activities of these enzymes may be explained by the phenomenon of diluted myocardium. This indicates that myocardial injury, as estimated from plasma enzyme activities, may still be expressed meaningfully in gram equivalents of healthy myocardium.

Quantitative analysis of plasma enzyme levels based upon simultaneous determination of different enzymes.

It is demonstrated that plasma elimination constants for rapidly eliminated circulating tissue enzymes can be obtained from plasma time-activity curves if a slowly eliminated reference enzyme is simultaneously sampled. Enzyme and reference enzyme must be released together into the plasma. From the elimination constants thus obtained enzyme release into the plasma can be calculated as a function of time. The method can be applied during continuous release of enzyme into the plasma. The validity of the method is tested in the dog by intravascular infusion of a preparation of cytoplasmic enzymes, obtained by incubating dog liver under anoxic conditions. Alanine aminotransferase (ALT) was used as reference enzyme. Infused quantities of aspartate aminotransferase (AST), glucosephosphate isomerase (GPI) and ALT can be estimated with coefficients of variation (CV) of respectively 10, 19 and 7.6%. Application of the method to plasma time-activity curves of enzymes in patients after acute myocardial infarction (AMI), with alpha-hydroxybutyrate dehydrogenase (HBD) as reference enzyme, results in the following values for the fractional catabolic rate constants (FCR) of AST, GPI, creatine kinase (CK) and its isoenzyme CK(MB): FCRAST = 0.093 +/- 0.006 h-1; FCRGPI = 0.27 +/ 0.03 h-1 (mean +/- SE, n = 14); FCRCK = 0.20 +/) 0.02 h-1 (n = 30); FCRCK(MB) = 0.34 +/- 0.08 h-1 (n = 16). These values are considerably higher than mentioned by most authors, and this indicates that enzyme release after AMI has been underestimated. After AMI, enzymes are released in quantities proportional to the enzyme content of human heart tissue. Average release of CK conforms to this rule but large variations for individual patients are observed. Accurate estimates of the quantities of enzymes released into the plasma can be made for slowly eliminated enzymes by the use of fixed mean values for elimination constants. The results presented to this study indicate that tissue enzymes released from infarcted myocardium in patients after AMI are recovered quantitatively in the plasma. Local inactivation of enzymes or inactivation during the transport from heart to plasma is not significant in such patients.

Liver damage as a potential source of error in the estimation of myocardial infarct size from plasma creatine kinase activity.

The occurrence of liver damage was investigated in patients with uncomplicated acute myocardial infarction (AMI). Cumulative plasma release of creatine kinase (CK) and alpha-hydroxybutyrate dehydrogenase (HBD) was compared with release of alanine aminotransferase (ALT). Up to 48 h after AMI, the appearance of ALT could be fully explained by myocardial ALT release. Thereafter additional release of ALT occurred, indicating liver damage. A possible effect of liver function on the rate of elimination of CK from plasma was studied in the dog. Complete temporary arrest of hepatic blood supply was obtained after previous implantation of a portacaval shunt, ligation of secondary inflows and blockade of retrograde perfusion. Neither these preliminary haemodynamic interventions nor the acute arrest of hepatic blood flow had any effect on the disappearance rate of CK from plasma. It is concluded that some liver damage commonly occurs in patients after AMI. However, this phenomenon does not interfere with the estimation of infarct size because the elimination of CK from plasma is unaltered during total hepatic ischaemia.

Measurement of myocardial infarct size from plasma fatty acid-binding protein or myoglobin, using individually estimated clearance rates.

OBJECTIVE: In patients with acute myocardial infarction (AMI), estimation of infarct size from the early markers, fatty acid-binding protein (FABP) and myoglobin (MYO), usually assumes average (fixed) rate constants (FCR) for protein clearance from plasma. However, individual variation in FCR is large. Renal dysfunction causes slower clearance of FABP and MYO from plasma and, hence, overestimation of infarct size in 20-25% of patients. We investigated whether or not more accurate values of infarct size could be obtained with individually estimated clearance rates. METHODS: Concentrations of FABP and MYO and, for comparison, activities of the established cardiac markers, creatine kinase (CK) and alpha-hydroxybutyrate dehydrogenase (HBDH), were assayed in serial plasma samples from 138 patients with AMI. Individual FCR values of FABP and MYO were estimated from plasma creatinine concentrations, sex and age. RESULTS: Individual FCR values varied from 0.4 to 2.4 h-1. Use of these individual FCR values significantly improved the correlation between infarct size, as estimated from FABP or MYO on the one hand, and from CK and HBDH on the other. Approximately equal estimates of infarct size were obtained for all four marker proteins. CONCLUSIONS: Using individually estimated clearance rates, renal insufficiency no longer hampers calculation of infarct size from FABP and MYO, and reliable estimates of total myocardial damage can be obtained within 24 h after first symptoms.

Estimation of circulatory parameters in patients with acute myocardial infarction. Significance for calculation of enzymatic infarct size.

Estimation of infarct size from enzyme activities in plasma or serum presupposes known values of circulatory parameters such as the extravascular distribution volume Ve and the permeability constant P for the transport of enzyme between intravascular volume Vi and Ve. In man, parameter values are used that are extrapolated either from values found in the dog or from turnover studies of non-myocardial proteins. Large systematic errors can be introduced in this way, as demonstrated in this study. It is shown that by simultaneous determination of two different enzymes in the same patient, estimates of circulatory parameters are obtained. The method is applied to creatine kinase (CK) and alpha-hydroxybutyrate dehydrogenase (HBDH) plasma activities in 36 patients with acute myocardial infarction (AMI). The following results are obtained: 1. Exchange of enzyme between Vi and Ve is much slower and clearance of CK is much faster than presently assumed in the literature. 2. Release of CK and HBDH into the circulation is proportional to the amounts of these enzymes present in the myocardium. This finding is supported by data on early enzyme release. 3. Quantitation of HBDH release needs a two-compartment model, while for CK a one-compartment model can be used in good approximation. 4. Release of CK and HBDH after AMI continues up to 96 hours. 5. Using obtained parameter values, a simulated model demonstrates that estimation of clearance rates of CK from exponential fits on plasma levels results in large errors. This may explain recent conflicting results in validation of enzymatic estimates of infarct size.

Peri-operative myocardial tissue injury and the release of inflammatory mediators in coronary artery bypass graft patients.

OBJECTIVE: This study was conducted to evaluate to what extent the ischemia-reperfusion injury resulting from the cardiopulmonary bypass (CPB) and aortic cross-clamping procedures during coronary artery bypass grafting (CABG) contributes to the systemic inflammatory response generally found in these patients. METHODS: Serum levels of enzymes (CK and CK-MB) and non-enzymatic proteins (FABP and myoglobin) as markers of myocardial tissue injury, bactericidal permeability increasing protein (BPI) as an indicator of neutrophil activation, interleukin-6 (IL-6) as inducer of the acute phase response and lipopolysaccharide binding protein (LBP) as parameter of the acute phase response were measured in 15 low-risk CABG patients with cardiopulmonary bypass (CPB), and 17 low-risk CABG patients without CPB. RESULTS: Already 0.5 h after reperfusion significantly increased plasma levels of all markers of myocardial tissue injury were noted in patients having surgery with CPB, but not in non-CPB patients. No significant differences were found between both groups for BPI and IL-6 levels in the early reperfusion period. BPI and IL-6 levels were higher in the non-CPB group on the first post-operative day (P < 0.05). However, no correlations were found for any marker of peri-operative tissue damage with either early neutrophil activation, or acute phase reactants. CONCLUSIONS: Perioperative myocardial injury resulting from CPB and aortic cross-clamping in low-risk CABG patients does not contribute to the release of inflammatory mediators in these patients.

Thrombolysis-induced coronary reperfusion causes acute and massive interstitial release of cardiac muscle cell proteins.

OBJECTIVE: Reperfusion of the infarct-related artery in patients with acute myocardial infarction limits infarct size, but also causes accelerated release into plasma of cardiac tissue proteins. The latter effect could reflect either enhanced protein washout from the heart or abrupt disruption of myocyte membranes. The present study indicates that the latter mechanism prevails. METHODS: In 26 patients, patency of the infarct-related artery was determined by coronary angiography 90 min and 5-7 days after thrombolytic treatment. Continuous electrocardiography was performed during the first 24 h after admission. Cumulative release of myoglobin (Mb) and creatine kinase (CK) into plasma was calculated from frequently sampled plasma concentrations. RESULTS: In patients with a patent infarct-related artery after 90 min, onset of a rapid (> 50%) decrease in ST-vector magnitude coincided with an equally rapid increase in QRS-vector magnitude, and with a sudden onset of release into plasma of Mb as well as CK. In these patients, a maximal initial release rate was observed and cumulative release conformed closely to a simple model for sudden interstitial liberation of proteins. In contrast, protein release started more gradually and could not be fitted to this model, in patients with persistent occlusion of the infarct-related artery at 90 min and absence of ST-vector normalisation. CONCLUSIONS: Previous studies have demonstrated significant myocardial salvage by timely reperfusion therapy. Nevertheless, this study indicates that the moment of recanalisation of the infarct-related artery coincides with sudden and massive disruption of myocyte membranes. Attenuation of this effect, if possible, could further improve the benefits of reperfusion therapy.

Adenoviral vector-mediated gene expression in the nervous system of immunocompetent Wistar and T cell-deficient nude rats: preferential survival of transduced astroglial cells in nude rats.

In the present paper, we examined the effect of the adenoviral vector dosage, the role of T cells, and the influence of the presence of replication-competent adenovirus (RCA) in adenoviral vector stocks, on the efficacy of adenoviral vector-directed transgene expression in the facial nucleus of immunocompetent Wistar and athymic nude rats. A small number of motor neurons and glial cells was transduced at low dosages of viral vector (1 x 10(6) pfu) and in the absence of RCA, and transgene-expressing cells persisted throughout the 3-week period of observation. Intraparenchymal infusion of 2 x 10(7) pfu of a recombinant adenoviral vector free of RCA was required for optimal transduction of facial motor neurons. In Wistar rats, a biphasic immune response occurred at higher dosages of the vector (5 x 10(6) and 2 x 10(7) pfu) that was characterized by early infiltration of macrophages and the occurrence of T cells during the second week after injection of the vector. The immune response was associated with the loss of transduced neural cells. In nude rats, administration of an adenoviral vector free of RCA resulted in a macrophage response comparable to that in the Wistar rat and long-term survival of transduced astroglial cells. However, transduced motor neurons degenerated according to a similar time course as observed in Wistar rats. Small amounts of RCA (2 x 10(5) pfu) injected with 2 x 10(7) pfu recombinant viral vector particles resulted in an accelerated T cell response and a rapid elimination of transduced cells within 1 week in Wistar rats, whereas in nude rats transgene expression continued during this period. Taken together, these observations suggest that at the high viral vector loads necessary to achieve optimal transduction of the facial nucleus, T cells play a role in the degeneration of adenoviral vector-transduced astroglial cells. The adverse effects on neurons appear to be due to the observed inflammatory response or to direct adenoviral vector toxicity.

Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system.

Recombinant adeno-associated virus (rAAV) vectors have become attractive tools for in vivo gene transfer. The production and purification of high-titer rAAV vector stocks for experimental and therapeutic gene transfer continue to undergo improvement. Standard rAAV vector purification protocols include the purification of the vector by cesium chloride (CsCl)-density gradient centrifugation followed by extensive desalination via dialysis against a physiological buffer for in vivo use. These procedures are extremely time consuming and frequently result in a substantial loss of the infectious vector titer. As an alternative to CsCl we have investigated the use of Iodixanol, an X-ray contrast solution, as the density-gradient medium. Purification of rAAV vectors by Iodixanol shortened the centrifugation period to 3 hr and resulted in reproducible concentration and purification of rAAV-vector stocks. We show that injection of rAAV derived from an Iodixanol gradient can be used for in vivo gene transfer applications in the brain and spinal cord without detectable cytopathic effects and directing stable transgene expression for at least 2 months.

Annexin V perturbs or stabilises phospholipid membranes in a calcium-dependent manner.

The potency of annexin V to transport Ca2+ ions across phospholipid membranes was investigated, using large unilamellar phospholipid vesicles loaded with the Ca2+ indicator fura-2. It was demonstrated that annexin V leaves the vesicle membranes intact when added in the presence of 1 mM Ca2+. However, if the vesicles were first incubated with annexin V in the absence of Ca2+, subsequent addition of Ca2+ produced a fluorescence signal due to binding of Ca2+ to fura-2. Centrifugation of the vesicle suspension immediately thereafter showed that this signal originated from the supernatant and not from the sedimented vesicles. Our results show that annexin V causes loss of vesicle integrity in the absence of Ca2+, and leakage of trapped fura-2, rather than inward Ca2+ transport. Bovine serum albumin or Ca2+ concentrations higher than 2.5 mM also caused such fura-2 leakage. Apparently, calcium-dependent binding of annexin V to the membrane prevents aspecific membrane damage caused by this protein.

Partial coverage of phospholipid model membranes with annexin V may completely inhibit their degradation by phospholipase A2.

Phospholipase A2 (PLA2)-mediated hydrolysis of membrane phospholipids was measured by ellipsometry, and the inhibition of this process by annexin V was studied. Planar membranes, consisting of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (PC/PE/PS; 54:33:13, on molar basis), were degraded by pancreatic PLA2, and the rate of hydrolysis was limited to about 0.7%/min. The influence of graded coverage of the membrane with annexin V was studied. The degree of PLA2 inhibition was nonlinearly related to the amount of membrane-bound annexin V, and binding of only 12% and 54% of full membrane coverage resulted in, respectively, 50% and 93% inhibition. These findings indicate that the inhibition of PLA2-mediated hydrolysis by annexin V cannot be simply explained by shielding of phospholipid substrates from the enzyme. Moreover, the present results leave room for a role of endogenous annexin V in regulating phospholipid turnover in the plasma membrane of parenchymal cells such as cardiomyocytes.

A continuous displacement immunoassay for human heart-type fatty acid-binding protein in plasma.

Human heart-type fatty acid-binding protein (FABP) is suggested as an early plasma marker of acute myocardial infarction (AMI), and several studies have proved that, for early diagnosis of AMI, FABP performs better than myoglobin, which is a more often used early marker protein. Because serial measurement of biochemical markers in plasma is now universally accepted as an important determinant in AMI diagnosis, a rapid and continuous measuring method for FABP would be desirable. The aim of the present study was to develop an immunoassay based on the principle of displacement and using a column for rapid and continuous measurement of FABP in plasma. Glass columns filled with Sepharose-bound FABP were loaded with a horseradish peroxidase (HRP)-labeled antibody (Ab) and equilibrated with human plasma. After reaching a stable baseline, human plasma spiked with FABP or plasma from AMI patients was added. The Ab-HRP complex dissociated due to the presence of FABP in the plasma and was subsequently quantified. For plasma from AMI patients (n=5), the Ab-HRP level thus measured correlated with the corresponding plasma FABP concentration (R=0.96). The results of this study show the feasibility of a sensor for continuous monitoring of FABP in plasma.

The influence of the thyroid function on the metabolic rate of prothrombin, factor VII, and factor X in the rat.

The influence of the thyroid function on the metabolic rate of prothrombin, factor VII, and X was studied in the rat. Disappearance rates of the three coagulation factors were measured after synthesis had been blocked with appropriate doses of warfarin, and reappearance rates were assessed upon induction of synthesis by high doses of vitamin K1 injected into rats displaying coumarin induced hypocoagulability. No statistically significant difference in the disappearance and production rates of any of the factors could be found between normal euthyroid rats and thyroxin-treated hypothyroid rats proven to be euthyroid. The differences between the two euthyroid groups and the hypothyroid group were highly significant, however: hypothyroidism results in an approximately 50% decrease of the metabolic rates of the three coagulation factors under study. The reappearance of the three factors, under euthyroid as well as hypothyroid conditions, showed a biphasic pattern: in the first two hours after vitamin K1 administration to warfarin treated rats, a rapid reappearance was observed, to the same extent for all three factors, in hypo- as well as euthyroid rats. This finding suggests that in vitamin K1 deficiency an intracellular accumulation of precursor proteins (PIVKAs) occurs, which after rapid conversion into biologically active coagulation factors by vitamin K1 are shed into circulation. The subsequent phase of reappearance is much slower and reflects the synthesis rate of coagulation enzymes. It is characteristic for each factor and clearly slower in hypothyroid rats than in euthyroid rats. From this an influence of thyroid function on the synthesis rate of the protein moiety of coagulation factors can be inferred. An apparent difference between disappearance and reappearance rate of the coagulation factors in the plasma, particularly pronounced for factors VII and X in euthyroid rats, could theoretically be explained as the consequence of the model used for derivation of these rates.