Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C

The gene coding for human thrombomodulin, a thrombin receptor on endothelial cells and a cofactor for the activation of anticoagulant protein C zymogen, was isolated from a human genomic library by employing human thrombomodulin cDNA as a probe. The nucleotide sequences of the gene and the adjacent 5' and 3' flanking regions were then determined. The nucleotide sequence of this gene with approximately 3.7 kilobase pairs was identical to that of the cDNA, indicating that the gene for human thrombomodulin is free of introns. Hybridization data showed that there is only a single thrombomodulin gene in the human genome.     

Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues

Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomodulin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had Mr approximately 100,000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar Kd's for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The Kd for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of 125I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir53-64 and the thrombomodulin fifth-EGF-domain peptide Tm426-444 displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV30-43 which is similar in composition and charge to Hir53-64 showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site.     

Decreased thrombin affinity of cell-surface thrombomodulin following treatment of cultured endothelial cells with beta-D-xyloside

Thrombomodulin, an endothelial cell-surface anticoagulant, has been postulated to contain a glycosaminoglycan. Thrombomodulin function was therefore studied in endothelial cells treated with beta-D-xyloside, an inhibitor of glycosaminoglycan attachment to proteoglycan core proteins. Beta-D-xyloside caused a reproducible 3 to 5-fold increase in the Km of thrombomodulin for thrombin and a 20-30% decrease in the rate of protein C activation by the thrombin-thrombomodulin complex. These results support a role for glycosaminoglycans in thrombomodulin function and suggest that beta-D-xylosides can be used to investigate both the anticoagulant mechanisms and the biosynthesis of cell-surface thrombomodulin.     

Activated protein C--an anticoagulant that does more than stop clots

Activated protein C (APC) is a glycoprotein derived from its precursor, protein C and formed by the cleavage of an activation peptide by thrombin bound to thrombomodulin. Originally thought to be synthesized exclusively by the liver, recent reports have shown that protein C is synthesized by endothelial cells, keratinocytes and some hematopoietic cells.APC functions as a physiological anticoagulant with cytoprotective, anti-inflammatory and anti-apoptotic properties. In vitro and preclinical data have revealed that APC exerts its protective effects via an intriguing mechanism requiring endothelial protein C receptor and the thrombin receptor, protease-activated receptor-1. Remarkably, even though APC cleaves this receptor in an identical fashion to thrombin, it exerts opposing effects.Recently approved as a therapeutic agent for severe sepsis, APC is now emerging as a potential treatment for a number of autoimmune and inflammatory diseases including lung disorders, spinal cord injury and chronic wounds. The future pharmacologic use of APC holds remarkable promise.     

The structure and function of mouse thrombomodulin. Phorbol myristate acetate stimulates degradation and synthesis of thrombomodulin without affecting mRNA levels in hemangioma cells

Thrombomodulin is an endothelial membrane anticoagulant protein that is a cofactor for protein C activation. We have evaluated the expression of thrombomodulin in cultured mouse hemangioma cells before and after treatment with phorbol myristate acetate (PMA), an agent that stimulates protein kinase C. We also isolated a cDNA encoding 481 amino acids of mouse thrombomodulin and the entire 3'-untranslated portion of its mRNA. The deduced amino acid sequence of mouse thrombomodulin is similar to those determined for human and bovine thrombomodulin. An S1 nuclease protection assay was used to measure thrombomodulin mRNA in hemangioma cells. The half-life for thrombomodulin mRNA was 8.9 +/- 1.8 h (S.D.) in cells treated with actinomycin D. Treatment with PMA had no effect on thrombomodulin mRNA levels. Thrombomodulin turnover was evaluated by immunoprecipitation of [35S]methionine-labeled thrombomodulin. The t1/2 was 19.8 +/- 3.9 h (S.D.); PMA treatment decreased the t1/2 to 10.9 +/- 1.1 h (S.D.) while increasing the rate of synthesis to a maximum of 190% of control. Protein C cofactor activity on hemangioma cells was reduced 35 +/- 4% by treatment with PMA within 30 min. This decrease was associated with a parallel decline in cell surface thrombomodulin antigen and with enhanced phosphorylation of thrombomodulin on serine residues. We conclude that thrombomodulin is phosphorylated in response to treatment of hemangioma cells with PMA which leads to decreased protein C cofactor activity and both increased degradation and synthesis of thrombomodulin.     

The endothelial cell protein C receptor (EPCR) functions as a primary receptor for protein C activation on endothelial cells in arteries, veins, and capillaries.

Plasma protein C functions as an anticoagulant when it is converted to the active form of serine protease. Protein C activation has been found to be mediated by the endothelial cell surface thrombin/thrombomodulin (TM) complex. In addition, we recently identified the endothelial cell protein C/activated protein C receptor (EPCR) which is capable of high-affinity binding for protein C. In this study, we established monoclonal antibodies (mAbs) against EPCR including several function blocking antibodies. Immunohistochemical analysis using these mAbs demonstrated that EPCR is widely expressed in the endothelial cells of arteries, veins, and capillaries in the lung, heart, and skin. Function blocking anti-EPCR mAbs strongly inhibited protein C activation mediated by primary cultured arterial endothelial cells which express abundant EPCR. Anti-EPCR mAbs also prevent protein C activation mediated by microvascular endothelial cells. These results indicate that EPCR functions as an important regulator for the protein C pathway in various types of vessels.     

The turnover of thrombin-thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. Endocytosis and degradation of thrombin

We have prepared a monoclonal antibody directed against human thrombomodulin. We used the antibody to measure thrombomodulin molecules in cultured human endothelial cells from umbilical vein and in a human lung cancer cell line (A549). Endothelial cells contain approximately 30,000-55,000 molecules of thrombomodulin/cell while the A549 cell has about 1/4 of this number. About 50-60% of thrombin binding sites on endothelial cells are thrombomodulin, while about 90% of thrombin binding sites on A549 cells are thrombomodulin. Exposure of these cells to thrombin decreased thrombomodulin on the cell surface suggesting that internalization of thrombin-thrombomodulin occurred. The internalized 125I-thrombin was degraded in the cells and thrombomodulin reappeared on the cell surface after 30 min, suggesting the recycling of thrombomodulin. The rate of protein C activation correlated with the presence of the thrombin-thrombomodulin complex on the cell surface. The binding of thrombin to cell-surface thrombomodulin accelerates protein C activation; the subsequent internalization of the thrombin-thrombomodulin complex is associated with cessation of protein C activation. Therefore, endocytosis of thrombin-thrombomodulin may serve to control protein C activation. The uptake and degradation of thrombin bound to thrombomodulin may provide a mechanism for clearance of thrombin from the circulation.     

Microthrombomodulin. Residues 310-486 from the epidermal growth factor precursor homology domain of thrombomodulin will accelerate protein C activation

Thrombomodulin is the endothelial cell cofactor for thrombin-catalyzed activation of protein C. Recently, we isolated a 10-kDa thrombin binding fragment, CB3, from the epidermal growth factor precursor homology domain (epidermal growth factor (EGF)-like regions) of thrombomodulin (Kurasawa, S., Stearns, D. J., Jackson, K.W., and Esmon, C.T. (1988) J. Biol. Chem. 263, 5993-5996). The CB3 fragment did not, however, support protein C activation. A 29-kDa fragment, called CB23, has now been isolated and corresponds to residues 310-486 in the EGF-like region of thrombomodulin. The CB23 fragment bound thrombin and accelerated thrombin-catalyzed protein C activation. With two separate preparations of CB23, the Km for protein C was 1.6 and 1.9 microM and the Kd for thrombin was 8.9 and 13.2 nM. The carboxyl terminus of CB23 and CB3 was identified by isolation and sequence analysis of a tryptic peptide from CB3. The sequence of this peptide corresponded to Asn457-Ser486, indicating that the carboxyl terminus of these fragments is 6 residues beyond the sixth EGF-like region of thrombomodulin. In addition, although CB3 cannot accelerate protein C activation, CB3 did inhibit the rate of thrombin-catalyzed fibrinopeptide release from fibrinogen. Thus, like native thrombomodulin, CB3 will alter thrombin's substrate specificity, but protein C activation requires additional information all of which can be provided by other regions of the EGF-like domain.     

Circulating lupus-type anticoagulant, a risk factor for thrombosis by inhibition of protein C activation

The lupus anticoagulant is a risk factor of thrombosis. The non thrombogenic endothelial surface could be a target for the lupus anticoagulant. We have investigated the effect of purified immunoglobulins G of five patients with LA on the thrombomodulin activity of cultured human endothelial cells from umbilical cord vein. The rate of activation of purified protein C (PC) (30 nM) by the endothelial cells in the presence of thrombin (0.1 U/dish) has been measured by hydrolysis of substrate S 2366. Activated PC has been 7.37 +/- 0.78 pmoles X ml-1 X h-1 in the presence of buffer and 7.2 +/- 0.78 pmoles X ml-1 X h-1 in the presence of control IgG (2 mg/dish). Heat aggregated IgG did not induce any significant change. Patient's IgG lowered significantly the rate of PC activation (4.86 +/- 1.04 pmoles X ml-1 X h-1, p less than 0.001). Fab fragment from two of these patient's IgG displayed the same inhibition. Moreover neutralization of this effect was obtained by addition of phospholipids (70% phosphatidylcholine, 30% phosphatidylserine) in excess to patient's IgG. Activation of PC has been also performed using purified rabbit thrombomodulin and a similar inhibition by patient's IgG was found. These results seem to indicate that antibodies present in the IgG fractions containing LA could be directed against phospholipids associated to thrombomodulin activity. Reduction of PC activation if present in the patients with LA could play a role in the occurrence of thrombosis.     

Ligand specificity of human thrombomodulin. Equilibrium binding of human thrombin, meizothrombin, and factor Xa to recombinant thrombomodulin.

Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.     

Relationship between anticoagulant activities and polyanionic properties of rabbit thrombomodulin

Rabbit thrombomodulin displays three distinct blood anticoagulant activities: it promotes the activation of protein C by thrombin (protein C activation cofactor activity); it promotes the inactivation of thrombin by thrombin (direct anticoagulant activity). The effects on these activities of mouse anti-thrombomodulin monoclonal antibodies and of the heparin-neutralizing proteins, platelet factor 4, histidine-rich glycoprotein, and S-protein, were investigated. One of the antibodies, which did not influence the functional properties of thrombomodulin, was used as an immunoaffinity ligand for purification of the protein. Two other antibodies, which were found to abrogate the protein C activation cofactor activity of the purified thrombomodulin, also abolished the antithrombin-dependent and the direct anticoagulant activities. The heparin-neutralizing proteins all inhibited the two latter activities, albeit to a varying extent, but did not appreciably affect the activation of protein C. These results are interpreted in relation to our previous finding that rabbit thrombomodulin contains an acidic domain, tentatively identified as a sulfated glycosaminoglycan (Bourin, M.-C., Boffa, M.-C., Bj?rk, I., and Lindahl, U. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5924-5928). It is proposed that the acidic domain interacts with thrombin at the protein C activation site and that this interaction is a prerequisite to the expression of direct as well as antithrombin-dependent anticoagulant activity. The interaction is not essential to, but compatible with, the activation of protein C. Experiments involving treatment of thrombomodulin with various glycanases or with nitrous acid, followed by measurement of anticoagulant activities, indicated that the acidic domain is constituted by a sulfated galactosaminoglycan and not by a heparin-related polysaccharide as previously suggested.     

Inactivation of chicken liver pyruvate carboxylase by 1,10-phenanthroline.

Human protein C is the precursor of a serine proteinase in plasma which contains nine 4-carboxyglutamic acid residues and functions as a potent anticoagulant. It is activated by thrombin in the presence of an essential endothelial-cell-membrane glycoprotein cofactor, thrombomodulin. In a purified human system, vitamin K-dependent proteins such as factor X, prothrombin and prothrombin fragment 1 were able to inhibit protein C activation by the thrombin-thrombomodulin complex, using either detergent-solubilized thrombomodulin or thrombomodulin reconstituted into vesicles consisting of phosphatidylcholine and phosphatidylserine (1:1, w/w). Factors VII and IX and protein S were much less efficient. Prothrombin fragment 1 behaved as a non-competitive inhibitor with apparent Ki values of 4 microM in the absence, and of 2-2.5 microM in the presence, of phospholipids. Heat decarboxylation of fragment 1 abolished its ability to interfere in protein C activation, and high phospholipid concentrations could attenuate its inhibitory effect and were responsible for a gradual loss of the non-competitive character. Fragment 1 also inhibited the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues, without any influence from phospholipids. At high thrombin concentrations, with respect to thrombomodulin, the inhibitory effect of fragment 1 was diminished. Fragment 1, at 3.8 microM, inhibited by 50% the activation of protein C (0.1 or 0.3 microM) by thrombin. These results suggest that the 4-carboxyglutamic acid domain of vitamin K-dependent proteins can act as a modulator of the protein C anticoagulant pathway through two distinct types of interaction. The functional 4-carboxyglutamic acid domain would be necessary to allow the enhancement of protein C activation in the presence of anionic phospholipids and it could recognize a phospholipid-independent binding site on the thrombin-thrombomodulin complex.     

Stable expression of a secretable deletion mutant of recombinant human thrombomodulin in mammalian cells

Thrombomodulin is an endothelial cell membrane protein which plays a central regulatory role in the protein C anticoagulant pathway. The human thrombomodulin intronless gene was isolated from a genomic DNA library and used to isolate the coding region. A mammalian expression vector, phd-TMD1, encoding all the extracellular domains of human thrombomodulin but lacking the transmembrane and cytoplasmic domains was constructed. Stable phd-TMD 1 transformants, in both hamster AV12-644 and human 293 cells, expressed functionally active recombinant thrombomodulin as a secreted, soluble product. Soluble thrombomodulin was secreted as two major proteins of 105 kDa and 75 kDa, both of which were purified to homogeneity. The kinetic properties for protein C activation of the two proteins were very different: the Kd for thrombin, Km for protein C, and Ca2+ optima were 3.0 nM, 1.5 microM, and 1-3 mM for the 105-kDa protein and 16 nM, 2.3 microM, and 0.2-0.5 mM for the 75-kDa protein. In clotting and platelet activation assays, the 105-kDa protein was a much more potent anticoagulant than the 75-kDa protein. Both forms of the protein had the amino-terminal sequence Ala19-Pro-Ala-Glu-Pro-Gln. Amino acid composition analysis indicated that both forms of the protein had the same amino acid content which was consistent with the predicted protein comprising residues Ala19 to Ser515. The difference in size appeared to be due to glycosylation as both forms were of similar size following chemical deglycosylation. These studies suggest that (1) secretable thrombomodulin derivatives can be used to study structure-function relationships of the extracellular domains of this important regulatory protein, (2) the extent of glycosylation has profound effects on the kinetic and anticoagulant properties of human thrombomodulin, and (3) soluble recombinant human thrombomodulins may be developed as clinically significant therapeutic anticoagulants.     

Tissue-restricted expression of thrombomodulin in the placenta rescues thrombomodulin-deficient mice from early lethality and reveals a secondary developmental block

The endothelial cell surface receptor thrombomodulin (TM) inhibits blood coagulation by forming a complex with thrombin, which then converts protein C into the natural anticoagulant, activated protein C. In mice, a loss of TM function causes embryonic lethality at day 8.5 p.c. (post coitum) before establishment of a functional cardiovascular system. At this developmental stage, TM is expressed in the developing vasculature of the embryo proper, as well as in non-endothelial cells of the early placenta, giant trophoblast and parietal endoderm. Here, we show that reconstitution of TM expression in extraembryonic tissue by aggregation of tetraploid wild-type embryos with TM-null embryonic stem cells rescues TM-null embryos from early lethality. TM-null tetraploid embryos develop normally during midgestation, but encounter a secondary developmental block between days 12.5 and 16.5 p.c. Embryos lacking TM develop lethal consumptive coagulopathy during this period, and no live embryos are retrieved at term. Morphogenesis of embryonic blood vessels and other organs appears normal before E15. These findings demonstrate a dual role of TM in development, and that a loss of TM function disrupts mouse embryogenesis at two different stages. These two functions of TM are exerted in two distinct tissues: expression of TM in non-endothelial extraembryonic tissues is required for proper function of the early placenta, while the absence of TM from embryonic blood vessel endothelium causes lethal consumptive coagulopathy.     

A domain composed of epidermal growth factor-like structures of human thrombomodulin is essential for thrombin binding and for protein C activation

Thrombomodulin, an endothelial thrombin receptor, acts as a cofactor for the thrombin-catalyzed activation of anticoagulant protein C. The extracellular region of human thrombomodulin consists of three tentative domains, a NH2-terminal domain (D1), a domain involving six consecutive epidermal growth factor-like structures (D2), and an O-glycosylation-rich domain (D3). To identify the domain onto which thrombin binds, a series of recombinant proteins corresponding to the entire protein, D1, D2, D1 + D2, D1 + D2 + D3, and D2 + D3 were expressed in simian COS-1 cells. The proteins were partially purified by rabbit anti-thrombomodulin-F(ab')2-agarose chromatography. Western blotting analysis showed the expression of the respective recombinant proteins. All proteins involving D2, as well as D2 alone, had cofactor activity that allowed binding directly to thrombin, but D1 did not. The cofactor activity of the entire protein but not the mutants is increased in the presence of phospholipids and this is the only protein that binds to the phospholipid layer. These results indicate that the domain involving the epidermal growth factor-like structures of thrombomodulin is essential for thrombin binding and expression of the cofactor activity for protein C activation and that none of the extracellular domains interact with phospholipids.     

Stability of the thrombin-thrombomodulin complex on the surface of endothelial cells from human saphenous vein or from the cell line EA.hy 926.

Protein C activation by alpha-thrombin on the surface of endothelial cells depends on an essential membrane-glycoprotein cofactor, thrombomodulin. In the present study we have monitored the activity of thrombin-thrombomodulin complexes on human saphenous-vein endothelial cells (HSVEC) or on the endothelial cell line EA.hy 926. Cell monolayers were exposed for 5 min to 8.5 nM human alpha-thrombin and then washed to remove unbound thrombin. The cells were then incubated at 37 degrees C for 5-180 min. At the end of the respective incubation periods, purified human protein C (120 nM) was added in order to assay the activity of the thrombin-thrombomodulin complexes present on the cell surface. HSVEC pre-exposed to thrombin retained their full capacity to promote protein C activation up to 90 min after free thrombin was removed. This capacity then decreased slowly to reach 56% of control value after 180 min of incubation. Original activity was 3.8 +/- 0.9 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 5). The capacity of protein C activation of EA.hy 926 cells remained constant for 120 min after free thrombin was removed, then decreased to 76% of control after 180 min. Original activity was 2.0 +/- 0.4 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 3). Similar results were obtained with cells fixed with 3% paraformaldehyde. However, during the 5-180 min incubation period, non-fixed cells of both types were capable of significantly internalizing fluorescent acetylated low-density lipoprotein. In the experimental protocol used here, an eventual inhibition of thrombin internalization by protein C can be excluded, as protein C is only added at the end of the incubation period. We conclude that there is no evidence of rapid internalization of thrombin-thrombomodulin complexes on HSVEC or the EA.hy 926 cell line, as assessed by the ability of membrane-bound thrombin to activate protein C.     

The effect of bovine thrombomodulin on the specificity of bovine thrombin

Bovine lung thrombomodulin is purified and used to investigate the basis of the change in substrate specificity of bovine thrombin when bound to thrombomodulin. Bovine thrombomodulin is a single polypeptide having an apparent molecular weight of 84,000 and associates with thrombin with high affinity and rapid equilibrium, to act as a potent cofactor for protein C activation and antagonist of reactions of thrombin with fibrinogen, heparin cofactor 2, and hirudin. Bovine thrombomodulin inhibits the clotting activity of thrombin with Kd less than 2.5 nM. Kinetic analysis of the effect of bovine thrombomodulin on fibrinopeptide A hydrolysis by thrombin indicates competitive inhibition with Kis = 0.5 nM. The active site of thrombin is little perturbed by thrombomodulin, as tosyl-Gly-Pro-Arg-p-nitroanilide hydrolysis and inhibition by antithrombin III are unaffected. Insensitivity of the reaction with antithrombin III is likewise observed with thrombin bound to thrombomodulin on intact endothelium. Antithrombin III-heparin, human heparin cofactor 2, and hirudin inhibit thrombin-thrombomodulin more slowly than thrombin. These effects may arise from a decrease in Ki of the inhibitors for thrombin-thrombomodulin or from changes in the active site not detected by tosyl-Gly-Pro-Arg-p-nitroanilide or antithrombin III. Bovine prothrombin fragment 2 inhibits thrombin clotting activity (Kd less than 7.5 microM) and acts as a competitive inhibitor of protein C activation (Kis = 2.1 microM). The data are consistent with a mechanism whereby thrombomodulin alters thrombin specificity by either binding to or allosterically altering a site on thrombin distinct from the catalytic center required for binding or steric accommodation of fibrinogen, prothrombin fragment 2, heparin cofactor 2, and hirudin.     

Isolation and characterization of thrombomodulin from human placenta

Protein C, a plasma protein, is activated by thrombin to a protease (protein Ca) that functions as a physiological anticoagulant. We have isolated thrombomodulin, a cofactor required for the rapid activation of protein C, from human placenta. The purification to near homogeneity was achieved using a crude Triton-solubilized protein fraction from a placental particulate fraction as starting material. Chromatography on DEAE-Sepharose removed 95% of the protein and achieved a 3-fold purification. Thrombomodulin was then isolated by affinity chromatography on a column of thrombin-Sepharose wherein the thrombin had been previously inactivated with diisopropyl fluorophosphate. The final preparation was purified 7,900-fold over the membrane extract with a yield of 7%. We obtained 0.88 mg of thrombomodulin from 100 g of membrane extract derived from 5 kg of placenta. The protein was nearly homogeneous as judged by electrophoresis on 10% acrylamide sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol with an apparent Mr = 105,000. Western blot analysis without 2-mercaptoethanol gave an apparent Mr = 75,000. The protein stimulated the rate of protein C activation by thrombin 800-fold to 10 mol of Ca formed/min/mol of thrombin. Thrombin and thrombomodulin appear to form a 1:1 stoichiometric complex as judged from experiments where we measured the effect of varying the concentration of thrombomodulin with respect to thrombin and the converse, on rates of protein C activation. An antibody directed against rabbit lung thrombomodulin inhibited the human placenta protein by 66%, and the amino acid composition of the proteins from the two species was similar indicating that the proteins are closely related. The apparent Michaelis constant of the thrombin-thrombomodulin complex for protein C is 9.8 microM. The protein C activation reaction requires calcium ions and is maximal at 1 mM Ca2+; higher concentrations inhibited the reaction. Coagulation factor Va and factor Va light chain both stimulate the activity of human thrombomodulin 2- to 3-fold.