Fast functional protein C assay using Protac, a novel protein C activator

A simple and rapid clotting method for the quantitative determination of protein C (PC) in plasma consists of the conversion of PC into activated PC (APC) by means of Protac, an activator protein isolated from Agkistrodon contortrix contortrix venom, of the subsequent degradation of factors V and VIII in PC immuno-depleted plasma by the generated APC and of the measurement of the prolongation of the activated partial thromboplastin time (APTT) which is proportional to the amount of PC in the sample. In 33 normal individuals a mean PC level of 97.1% of a normal pooled plasma was found. Comparison with an enzyme-immunoassay for PC in 33 patients with liver disease revealed a good correlation (r = 0.986). Patients under warfarin therapy (n = 34) had a mean PC level of 19.8%; a comparison with the immunological assay (mean value = 55.3%) in the same population suggested that the assay did not co-estimate acarboxy forms of PC. The assay proved to be insensitive to heparin concentration lower than 1 U/ml. Due to its simplicity, it should be suitable for diagnostic routine and monitoring of patients with abnormal PC level, even if under anticoagulation.     

Effect of protein C and activated protein C on coagulation and fibrinolysis in normal human subjects

Although protein C (PC) and activated protein C (APC) have been postulated to be useful for treating patients with thrombosis, their critical effect remains to be studied in human subjects. To examine whether purified PC or APC are useful for treating patients with thrombosis without showing any adverse effect, we studied effects on coagulation and fibrinolysis in normal human subjects. When highly purified human PC was administered intravenously to healthy subjects, plasma levels of immunoreactive PC decreased with a half-life of 10.9 h. Intravenously administered APC decreased with a half-life of 23 min as measured by prolongation of activated partial thromboplastin time (APTT). However, 1.7 h was obtained for the plasma half-life of APC when it was measured immunologically. These findings suggested that a significant fraction of the administered APC was rapidly inhibited by plasma inhibitor. Upon administration of APC, APTT was prolonged and plasma levels of clotting factor VIII (F-VIII) decreased transiently as measured by clotting assay. However, when determined by a chromogenic assay method in which 120-fold diluted plasma samples were used, plasma levels of F-VIII remained unchanged. Plasma levels of F-V did not decrease after APC administration. These findings suggested that prolongation of APTT and apparent decrease in plasma F-VIII clotting activity might be due to the in vitro-effect of APC present in plasma samples used. Diurnal fluctuation of plasminogen activator inhibitor in normal subject was not affected by administration of APC. Thus, PC or APC seems to function selectively at the site of thrombin-formation without lowering plasma levels of coagulation factors.     

Determination of functional and antigenic protein C inhibitor and its complexes with activated protein C in plasma by ELISA''s

Enzyme-linked immunosorbent assays (ELISA's) were developed for the measurement of protein C inhibitor (PCI) antigen and activity and for its complexes with activated protein C (APC) in plasma. For PCI activity and antigen, APC or anti-PCI, respectively, was immobilized to microtiter plates and PCI bound was detected with labelled anti-PCI antibodíes. For APC:PCI complexes, two different antibodies directed against protein C and PCI were used. The assays for PCI were calibrated with pooled normal human plasma (NHP) and with purified PCI, and for APC:PCI complexes with known concentrations of purified pre-formed complexes added to buffer or to plasma. The lower limit of sensitivity of the PCI activity and antigen assays was 10 ng/ml and 0.5 ng/ml, respectively and for plasma APC:PCI complexes 12 ng/ml. Mean coefficients of variation of 1.5 % to 5.8 % (intro-assay) and 2.1 % to 9.8 % (inter-assay) were found for the assays. For PCI antigen, a range of 56 % to 162 % of the NHP value was obtained in samples from 70 healthy donors (mean ±SD = 98.6 % ±23.1%). For PCI activity, the range was 59 % to 148 % (94.3 % ± 20.2). A good correlation (0.92) was obtained when both assays were compared. Plasma levels of APC:PCI complexes in 30 normals were under the detection limit (< 12 ng/ml). In plasma samples from 10 patients with disseminated intravascular coagulation (DIC) PCI antigen concentrations were decreased (55.6% ±20%) and 8 of the patients had APC-PCI complex levels between 32 and 240 ng/ml (median, 35 ng/ml). After addition of 20 ωg/ml APC to NHP or to protein C depleted plasma, 6.1 μg/ml complexes were recovered after 90 min incubation. Incubation of 10 μg/ml APC with NHP in the presence of 10 U/ml heparin yielded 11 μg/ml complexes after 90 min, which represent more than 90 % of the maximum possible value. Thus, the method should be adequate to study complexes of APC in vivo in clinical conditions in which activation of protein C pathway may occur.     

Interaction of plasma kallikrein with protein C inhibitor in purified mixtures and in plasma.

The interaction between plasma kallikrein (KK) and protein C inhibitor (PCI) and the influence of KK on the complex formation between activated protein C (APC) and PCI was studied in purified systems as well as in plasma in order to assess the significance of these reactions in the plasma milieu. PCI complexed to KK (KK:PCI) or to APC (APC:PCI) was measured by sandwich ELISA's using antibodies directed against each protein in the complexes. The formation of KK:PCI complexes assayed by this method paralleled the inhibition of KK amidolytic activity by PCI in purified system. Incubation of normal plasma (NHP) at 4 degrees C, which can induce prekallikrein activation due to cold activation, resulted in PCI inactivation and appearance of KK:PCI complexes. PCI activity fell to 35% of the NHP and 1.2 micrograms/ml of KK:PCI complex was formed. However, incubation of NHP at room temperature or of prekallikrein deficient plasma at 4 degrees C did not result in significant decrease of PCI activity. Thus the PCI inactivation was associated with prekallikrein activation and complexation to PCI following cold activation. Incubation of exogenous purified KK with NHP resulted in PCI inactivation and complexation with KK in a temperature-dependent manner. Addition of 2.8 micrograms/ml KK to plasma at 4 degrees C resulted in the inactivation of 55% of plasma PCI and the formation of 0.9 microgram/ml KK:PCI which represents 21% of the KK added, whereas at 37 degrees C PCI was inactivated to 30% and only 0.30 microgram/ml KK:PCI complexes were measured. These results indicate that PCI is a major KK inhibitor at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant human protein C: comparative functional studies with human plasma protein C

Protein C (PC) is the central protein in a major antithrombotic regulatory mechanism. Hereditary deficiencies of PC are associated with thrombosis. Therapeutic PC replacement may be an important treatment if pure functional human protein C is available in sufficient quantity. Human PC has been produced on a commercial scale using recombinant techniques. To study the functional properties of recombinant protein C (r-PC), we undertook a comparative investigation of the basic properties of r-PC and plasma protein C (n-PC). Both were isolated by immunopurification methods. Protac C activation proceeded at the same rate and kinetics for both forms. With thrombin-thrombomodulin (T-TM) activation, r-PC is significantly better than the activation of n-PC (for r-PC: Kcat/Km = 378 vs. n-PC: Kcat/Km = 35). No difference in the anticoagulant (aPTT prolongation) or profibrinolytic activities (inactivation of PAI-1 and PAI-3) were observed between activated r-PC and n-PC. Based on these functional studies, recombinant protein C has similar properties to the plasma form of protein C. However, T-TM activation of r-PC occurs faster than the n-PC. The mechanism is unknown, but may be due to the presence of larger amounts of single chain protein C which exists in a conformation more rapidly activated by the T-TM complex.     

Plasma protein S activity measured using Protac, a snake venom derived activator of protein C

Functional activity of protein S, a cofactor of activated protein C-dependent inhibition of blood coagulation, in human plasma was measured by using Protac, a snake venom derived activator of protein C. This assay appeared to be specific for protein S, because 1) the activated partial thromboplastin time of protein S-depleted plasma depended on the purified protein S added in the presence of Protac; and 2) the level of protein C in plasma sample (0 to 10 micrograms/ml) had no influence on the clotting time. The cofactor activity of protein S in the plasma of normal men (n = 16) and women (n = 14) was 99.4 +/- 23.8% and 98.6 +/- 24.5% respectively. The protein S activity in the plasma of pregnant women at pre- and post-partum (n = 14), and that in the plasma of patients under warfarin therapy (n = 20) were 46.2 +/- 18.9%, 45.8 +/- 19.6% and 24.0 +/- 15.7%, respectively. In these plasmas, the levels of protein S activity were lower than those of total protein S antigen, but were similar to those of free protein S antigen. In 16 patients out of two families with congenital protein S deficiency, the protein S activity, the free antigen and the total antigen were 9.4 +/- 6.9%, 13.3 +/- 4.6% and 57.4 +/- 20.7%, respectively. There was no significant relationship between the level of protein S activity and that of a complemental C4b-binding protein antigen in any of these patients.     

A simplified PTT-based protein C activity assay using the thrombin-thrombomodulin complex

A simplified assay for protein C activity in plasma is described which uses the ability of rabbit lung thrombomodulin to inhibit the procoagulant activity of thrombin while stimulating protein C activation. Barium eluates of plasma are activated for one hour at 37 degrees C by a mixture of human thrombin and rabbit lung thrombomodulin at concentrations which neutralize each other's effect on the kaolin-cephalin activated partial thromboplastin time (PTT). Protein C anticoagulant activity in the activated eluates is then measured directly in the PTT. The method is independent of protein S levels in the test samples, and is suitable for warfarinized and heparinized plasma. Protein C levels obtained with this method correlate closely with functional levels of vitamin K-dependent procoagulants as measured by the prothrombin and proconvertin time (P&P) in normal subjects and in patients receiving warfarin, indicating specificity for gamma-carboxylated protein C. The method has the potential to detect molecular variants defective in any of the interactions required for generation of anticoagulant activity in vivo.     

Determination of plasma protein S--the protein cofactor of activated protein C

Protein S, an important cofactor of activated protein C, and C4b-binding protein were purified from human plasma. Specific antibodies against the purified proteins were raised in rabbits and used for the development of immunologic assays for these proteins in plasma: an immunoradiometric assay for protein S (which measures both free protein S and protein S complexed with C4b-binding protein) and an electroimmunoassay for C4b-binding protein. Ranges for the concentrations of these proteins were established in healthy volunteers and patients using oral anticoagulant therapy. A slight decrease in protein S antigen was observed in patients with liver disease (0.78 +/- 0.25 U/ml); no significant decrease in protein S was observed in patients with DIC (0.95 +/- 0.25 U/ml). Criteria were developed for the laboratory diagnosis of an isolated protein S deficiency.     

Severe protein C deficiency in congenital thrombotic disease--description of an immunoenzymological assay for protein C determination

An immunoenzymatic assay (ELISA) is described for the quantitative assay of protein C in plasma. This technique allows a safe, reliable, and sensitive assay of protein C, and is easily used for routine investigation. Using this technique, a protein C deficiency (0.16 U/ml) was discovered in a 16 year old patient with a history of very severe thrombotic disease. Protein C deficiency was also discovered in his mother (0.62 U/ml) and father (0.50 U/ml). We therefore suggest that this case could represent a homozygous deficiency of protein C.     

Brain-specific protein C activation during carotid artery occlusion in humans

BACKGROUND AND PURPOSE: Activation of plasma protein C (PC) zymogen by thrombin-thrombomodulin at the endothelial surface is an important endogenous antithrombotic mechanism. It is unknown whether activated protein C (APC) is generated in vivo in the cerebrovasculature, because there is only limited thrombomodulin expression in human brain vascular endothelium. Therefore, we tested the hypothesis that carotid occlusion produces brain-specific PC activation. METHODS: Blood samples were simultaneously collected from the ipsilateral internal jugular vein and radial artery before and during carotid cross-clamping and on "de-occlusion" in 8 awake patients undergoing routine carotid endarterectomy. Plasma PC zymogen and circulating APC levels were measured using enzyme immunocapture assay and expressed as percent of pooled plasma controls. RESULTS: Internal jugular vein APC levels increased 28% exclusively during carotid occlusion and then decreased 32% with de-occlusion (F=8.1, P<0.005). PC zymogen increased only 5.9% with occlusion (F=6.3, P<0.02), consistent with hemoconcentration. There were no changes in radial artery PC or APC levels. CONCLUSIONS: These findings demonstrate brain-specific protein C activation in humans during carotid occlusion and suggest a protective role for endogenous APC generation during cerebrovascular occlusion.     

Platelets augment rabbit cerebral artery constriction by activating protein kinase C

BACKGROUND AND PURPOSE: We tested the hypothesis that activated platelets augment cerebral artery responsiveness to thrombin by activating protein kinase C, a possible intracellular modulator of Ca2+ sensitivity. METHODS: Ring segments of rabbit basilar artery were prepared for in vitro measurements of isometric force. Cumulative concentration-response curves to thrombin were made in the absence or presence of activated platelets. RESULTS: Arteries contracted to thrombin in a concentration-dependent manner; freshly obtained human platelets (8.5 x 10(8) cells/ml) activated with 10 micrograms/ml collagen increased the arteries' sensitivity to thrombin by threefold while augmenting the maximal response from 40 +/- 11% to 66 +/- 12% of the maximal response to 0.3 mM histamine. At 10 nM, staurosporine, an inhibitor of protein kinase C activity, blunted the platelet-induced augmentation of the response to thrombin but did not alter the sensitivity or the maximal contraction to 8-64 mM K+. Removal of the endothelium did not alter the characteristics of the thrombin concentration-response curve. The addition of activated platelets increased the tissue sensitivity of endothelium-denuded arteries to thrombin by a factor of 30, and the maximal response to thrombin was 101 +/- 27% of the histamine response. CONCLUSIONS: Our findings suggest that amplification of the arterial contractile response to thrombin caused by platelet-derived mediators may be due to activation of protein kinase C. There may also be a protective role for the cerebrovascular endothelium since endothelium reduced the extent of augmentation of protein kinase C-mediated tone by vasoactive agents released by activated platelets. Thus, increased vascular responsiveness caused by activated platelets may in part be due to protein kinase C-mediated changes in the intracellular sensitivity to Ca2+.     

Activation of human plasma prekallikrein: Influence of activators,activation time and temperature and inhibitors

Plasma prekallikrein (PKK) was activated with Cephotest and dextran sulphate (DS) under various conditions and the resulting kallikrein (KK) assayed on a chromogenic peptide substrate for this enzyme. Cephotest: plasma ratios of 30:1 and DS concentrations of between 0.001 and 0.01% (equal volumes plasma and DS) gave maximal activities when activation proceeded at 0°C. However, enzyme activity was also dependent on incubation time and temperature and activation times of 3 minutes (Cephotest) and 15 minutes (DS) at 0°C gave highest activities. Incubation at 37°C gave progressively lower KK yields with Cephotest and very little KK activity was detected with DS at this temperature. These results were shown to be due to progressive inhibition of plasma KK at 37°C. With C¹-esterase inhibitor deficient plasma higher KK activities were obtained with both activators at 0°C and the loss of activity on incubation at 37°C was markedly reduced. The addition of pure C¹-esterase inhibitor to the deficient plasma prior to activation resulted in a normal activity and normal inhibition profile. Gel filtration studies on normal and C¹-esterase deficient plasmas after activation at 0°C with Cephotest revealed that both free KK and KK-α₂ macroglobulin complexes were present in the activated plasmas. In the deficient plasma both activities were higher indicating that in normal plasma some KK is immediately bound by α₂-macroglobulin and C¹-esterase inhibitor even when activation takes place at 0°C.     

Protein C inhibitor: Purification and proteinase reactivity

Protein C inhibitor was purified from human plasma by a modification of a published procedure (Suzuki, K., Nishioka, J., and Hashimoto, S. J. Biol. Chem. 258, 163–168, 1983). Approximately 1 mg of pure protein was obtained from 1 L plasma, a yield of about 17%. The protein C inhibitor preparation did not lose activity over 4 weeks at 4°C. Second order rate constants were measured for the inhibition of activated protein C, thrombin, and urokinase, and bimolecular complexes of protein C inhibitor with activated protein C and thrombin were visualized by denaturing polyacrylamide gel electrophoresis. Heparin accelerated the inhibition of the three proteinases in a manner consistent with a template mechanism. Plasma or pure protein C inhibitor (at the same concentration) showed the same effect of heparin on activated protein C inhibition, indicating that protein C inhibitor accounts for all the heparin-dependent inhibition of activated protein C in vivo.     

Determination of plasma protein C inhibitor and of two activated protein C-inhibitor complexes in normals and in patients with intravascular coagulation and thrombotic disease

We developed an ELISA to quantitate complexes of activated protein C (APC) with a major plasma APC inhibitor, alpha 1-antitrypsin (alpha 1AT) in human plasma based on the sandwich principle using two different antibodies directed towards protein C and alpha 1AT, respectively. This ELISA test was specific for APC:alpha 1AT complexes and sensitive to greater than or equal to 150 pg complex. Fifty-one of 56 healthy donors had APC:alpha 1AT complex levels above the detection limit (3 ng/ml) ranging from 4 to 14 ng/ml (mean value +/- SD: 7.6 +/- 2.5 ng/ml). Patients (n = 10) with disseminated intravascular coagulation (DIC) had detectable levels of APC:alpha 1AT complex ranging from 21 to 125 ng/ml (median: 69 ng/ml). Complexes of APC with plasma protein C inhibitor (PCI) were also measured using an ELISA sandwich assay. None of the 30 healthy donors had detectable levels (greater than or equal to 5 ng/ml) of APC:PCI complex, and plasma samples from 9 of 10 DIC patients had detectable concentrations of APC:PCI complex ranging from 10 to 63 ng/ml (median: 22 ng/ml). APC:alpha 1AT complex was detected in 25 of 26 patients with deep venous thrombosis (DVT), with levels ranging from 5 to 136 ng/ml (median: 23 ng/ml), whereas APC:PCI was detected in only 6 DVT patients, with levels between 11 and 105 ng/ml. PCI antigen levels in 70 normals ranged from 56 to 175% (mean +/- SD: 99.1% +/- 24.2%). PCI antigen levels were decreased in DIC patients, in patients with cerebral arterial thrombosis, and in DVT patients undergoing heparin therapy, but not in patients with myocardial infarction. PCI antigen levels were decreased much further in DVT patients receiving heparin compared to those not receiving heparin, showing that heparin therapy is associated with a decrease in PCI levels. The detection in normal subjects and in thrombotic patients of circulating APC:inhibitor complexes supports the view that the protein C pathway is activated during DIC and DVT. Moreover, it emphasizes that both PCI and alpha 1AT are physiologic inhibitors of APC. Thus, measurement of APC complexes may provide sensitive parameters for specific detection of activation of the clotting and protein C pathways.     

A functional assay for protein C in human plasma

We describe a functional assay for protein C in human plasma samples based on the ability of activated protein C to prolong the kaolincephalin activated partial thromboplastin time of normal plasma. Protein C is separated from its inhibitor by elution of a barium citrate precipitate, and activated by incubation with human alpha-thrombin for one hour. Thrombin is then inhibited by antithrombin III and heparin, heparin neutralized by protamine sulfate, and protein C activity measured in the partial thromboplastin time. 24 normal subjects had a mean protein C level of 94 ± 12% (SD) of the activity in pooled normal plasma. Seven patients with severe liver disease had a mean protein C of 28%. Eleven patients with disseminated intravascular coagulation had a mean protein C of 29%. Eight patients receiving warfarin therapy had a mean protein C of 17%. The assay is relatively simple and should be suitable for general laboratory use.     

Sensitivity of the ProC global assay for protein C pathway abnormalities. clinical experience in 899 unselected patients with venous thromboembolism.

The ProC Global is a clotting assay designed to globally evaluate the functionality of the protein C (PC) pathway, which is found defective in up to 30% of the Caucasian patients with thrombophilia. It is based on the ability of endogenous activated protein C (APC), generated by activation of PC by a snake venom extract, to prolong an activated partial thromboplastin time (APTT). This retrospective study was carried out to evaluate the ability of this assay to distinguish patients with or without any PC pathway abnormalities in a cohort of 899 unselected patients with a history of thrombosis. The result of the ProC Global assay, expressed in PC activation time-normalized ratio (PCAT-NR), was significantly lower in patients in whom was previously demonstrated an abnormality of the PC pathway compared to those without. The cut-off level of PCAT-NR=0.75 was found to provide the best sensitivity-specificity ratio, since all the patients with the factor V (FV) Leiden mutation (n=71), APC resistance (n=3), PC deficiency (n=22), or combined defects (n=19) had a PCAT-NR below that value. The sensitivity of the ProC Global assay for a low protein S (PS) level (n=56) was only 66%, and was even weaker in the case of hereditary PS deficiency (46.6%, n=15). The assay did not perform well in samples from patients on oral anticoagulant treatment (OAT, n=64) or with liver failure (n=4), as the PCAT-NR was reduced in most cases, even in the absence of any abnormality. These results suggest that the ProC Global assay could be validly used as a first-step screening test for the FV Leiden-related APC resistance and PC deficiency in patients not on OAT. Given the moderate sensitivity of the assay for PS deficiency, this coagulation inhibitor must be determined in every case. However, the overall benefit of such a screening strategy is limited since more than 38% of the 659 patients without abnormality had a decreased PCAT-NR.     

Involvement of Lys 62(217) and Lys 63(218) of human anticoagulant protein C in heparin stimulation of inhibition by the protein C inhibitor.

Inhibition of activated protein C (APC) by protein C inhibitor (PCI) is stimulated by heparin, whereas inhibition by alpha1-antitrypsin (AAT) is heparin-independent. Three lysine residues located in a positively charged cluster in the serine protease domain of protein C (PC) were mutated to probe their involvement in the heparin stimulation of inhibition by PCI. These mutations were selected after analysis of the three-dimensional structure of APC and of molecular models for PCI and the APC-PCI complex. A double mutant, K62[217]N/K63[218]D, a single mutant, K86[241]S, and wild-type PC were expressed in embryonic human kidney 293 cells. Heparin stimulated the rate of inhibition of wt-APC by PCI approximately 400-fold, with second order rate constants (k2) in the absence and presence of heparin of 0.72 x 10(3) M(-1)s(-1) and 2.87 x 10(5) M(-1)s(-1), respectively. In contrast, heparin only yielded a 52-fold stimulation of the rate of inhibition of the double mutant APC by PCI as the rate constants in the absence and presence of heparin were k2 = 2.44 x 10(3) M(-1)s(-1) and k2 = 1.26 x 10(5)M(-1)s(-1), respectively. The double mutant K62N/K63D eluted at approximately 10% lower NaCl concentration from a heparin Sepharose column than the K86S mutant or wt-APC. These data suggest K62 and K63 in APC to be part of a heparin binding site which is important for heparin-mediated stimulation of inhibition of APC by PCI.     

Species specificity of the fibrinolytic effects of activated protein C.

Activated protein C (APC) has been shown to stimulate fibrinolysis in both in vitro and in vivo experimental systems. In order to test the importance of protein S in the fibrinolytic activity of APC we have compared the activity of APC, prepared from rabbit, bovine and human plasma, in the stimulation of whole blood clot lysis, the inactivation of plasminogen activator inhibitors and anticoagulant activity. When whole blood clot lysis was performed using tissue plasminogen activator in either human or rabbit blood, APC was found to enhance clot lysis in a species specific manner that paralleled the pattern observed for its anticoagulant activity. Bovine APC, was the poorest stimulator of fibrinolysis in human plasma. However, if bovine protein S was also added to human plasma, bovine APC was as effective in promoting fibrinolysis as human APC. In contrast, no species specific effects on the inactivation of plasminogen activator inhibitor activity was observed. Though substantial effects of APC on plasminogen activator inhibitor levels were made by rabbit, human and bovine activated protein C in human plasma, there was no effect of activated protein C on the rate of clot lysis of human plasma. These results suggest that protein S is important for the expression of the fibrinolytic activity of activated protein C and that the effect of protein S may be useful for the differentiation of fibrinolytic effects of activated protein C that may be related to the inactivation of plasminogen activator inhibitors and those that are not.     

Impairment of the protein C anticoagulant pathway in a patient with systemic lupus erythematosus, anticardiolipin antibodies and thrombosis

We have identified an inhibitor of the protein C anticoagulant pathway in the plasma of a patient with systemic lupus erythematosus and a history of recurrent deep vein thrombosis, fetal wastage, and seizures. The patient's plasma contained anticardiolipin antibodies as well as a weak lupus anticoagulant. Examination of this patient's plasma revealed normal levels of protein C and protein S antigen, normal levels of functional protein C, as well as essentially normal levels of every blood coagulation factor. In a modified prothrombin time assay, the activated protein C-mediated prolongation of the clotting time observed in normal plasma was not observed in this patient's plasma. Gel permeation chromatography of the patient's plasma revealed that the inhibitory material was a high molecular weight protein that coeluted with the IgM peak. The inhibitor did not appear to circulate as a complex with protein C, since the inhibitor could easily be separated from protein C during fractionation procedures, and did not interfere with the activation of protein C in plasma as assessed by a functional amidolytic assay. Our findings suggest that the recurrent thrombotic episodes observed in this patient may have occurred as a result of the patient's antiphospholipid antibody neutralizing specific phospholipids essential for the full expression of the anticoagulant activity of activated protein C.     

Thrombin inhibitors suppress the thrombin-thrombomodulin-mediated generation of activated protein C.

In the treatment of unstable coronary artery disease, direct thrombin inhibitors have shown no or only limited benefit as compared with heparin, despite theoretical advantages. One explanation may be that the direct thrombin inhibitors to a greater extent than heparin have an inhibiting effect on the generation and activity of activated protein C. In the present study, this hypothesis was tested in an in vitro, "purified" system, where human protein C underwent activation to activated protein C by the thrombin-thrombomodulin complex. Direct thrombin inhibitors, inogatran and hirudin, unfractionated heparin+antithrombin, or dalteparin+antithrombin, were added to the system before activation to evaluate their inhibitory effect on the generation of activated protein C. At inhibitor concentrations well below the achieved plasma levels in major clinical trials, the thrombin-thrombomodulin-mediated activation of protein C was inhibited by all the studied inhibitors in a dose-dependent manner, but, contrary to our hypothesis, to a greater extent by unfractionated heparin+antithrombin and dalteparin+antithrombin than by the direct thrombin inhibitors, hirudin and inogatran. Despite difficulties to draw conclusions for the in vivo situation, the in vitro inhibition, by all studied inhibitors, of the generation of activated protein C, found in this study may be a possible explanation for ongoing cardiovascular events despite adequate treatment with thrombin inhibitors, in patients with unstable coronary artery disease. This inhibition of the generation of activated protein C may also contribute to the rebound in cardiovascular events after withdrawal of effective antithrombotic treatment.