Ca2+ dependence of the interactions between protein C, thrombin, and the elastase fragment of thrombomodulin. Analysis by ultracentrifugation.

The two-way and three-way interactions among active-site-blocked bovine thrombin, bovine protein C, and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation at 23.3 degrees C in 100 mM NaCl, 50 mM Tris (pH 7.65), and 1 mM benzamidine, in the presence of 0 to 5 mM calcium chloride. Thrombin and elTM form a tight (Kd less than 10(-8) M) 1:1 complex in the absence of Ca2+ that weakens with the addition of Ca2+ (Kd approximately 4 microM in 5 mM Ca2+). Without Ca2+, thrombin and protein C form a 1:1 complex (Kd approximately 1 microM) and what appears to be a 1:2 thrombin-protein C complex. The Kd for the 1:1 complex weakens over 100-fold in 5 mM CaCl2. Protein C and elTM form a Ca(2+)-independent 1:1 complex (Kd approximately 80 microM). Nearly identical binding to thrombin and elTM is observed when active-site-blocked activated bovine protein C is substituted for protein C. Thrombin inhibited by diisopropyl fluorophosphate and thrombin inhibited by a tripeptide chloromethyl ketone exhibited identical behavior in binding experiments, suggesting that the accessibility of protein C to the substrate recognition cleft of these two forms of thrombin is nearly equal. Human protein C binds with lower affinity than bovine protein C. Ternary mixtures also were examined. Protein C, elTM, and thrombin form a 1:1:1 complex which dissociates with increasing [Ca2+]. In the absence of Ca2+, protein C binds to the elTM-thrombin complex with an apparent Kd approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of rabbit thrombomodulin into phospholipid vesicles

The influence of phospholipid on thrombin-thrombomodulin-catalyzed activation of protein C has been studied by incorporating thrombomodulin into vesicles by dialysis from octyl glucoside-phospholipid mixtures. Thrombomodulin was incorporated into vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (30% phosphatidylserine and 70% phosphatidylcholine). Thrombomodulin is randomly oriented in vesicles of different phospholipid composition. Incorporation of thrombomodulin into phosphatidylcholine, with or without phosphatidylserine, alters the Ca2+ concentration dependence of protein C activation. Soluble thrombomodulin showed a half-maximal rate of activation at 580 microM Ca2+, whereas half-maximal rates of activation of liposome-reconstituted thrombomodulin were obtained between 500 microM Ca2+ and 2 mM Ca2+, depending on the composition (protein:phospholipid) of the liposomes. The Ca2+ dependence of protein C activation fits a simple hyperbola for the soluble activator, while the Ca2+ dependence of the membrane-associated complex is distinctly sigmoidal with a Hill coefficient greater than 2.4. In contrast, the Ca2+ dependence of gamma-carboxyglutamic acid (Gla) domainless protein C activation is unchanged by membrane reconstitution (1/2 max = 53 +/- 10 microM) and fits a simple rectangular hyperbola. Incorporation of thrombomodulin into pure phosphatidylcholine vesicles reduces the Km for protein C from 7.6 +/- 2 to 0.7 +/- 0.2 microM. Increasing phosphatidylserine to 20% decreased the Km for protein C further to 0.1 +/- 0.02 microM. Membrane incorporation has no influence on the activation of protein C from which the Gla residues are removed proteolytically (Km = 6.4 +/- 0.5 microM). The Km for protein C observed on endothelial cells is more similar to the Km observed when thrombomodulin (TM) is incorporated into pure phosphatidylcholine vesicles than into negatively charged vesicles, suggesting that the protein C-binding site on endothelial cells does not involve negatively charged phospholipids. In support of this concept, we observed that prothrombin and fragment 1, which bind to negatively charged phospholipids, do not inhibit protein C activation on endothelial cells or TM incorporated into phosphatidylcholine vesicles, but do inhibit when TM is incorporated into phosphatidylcholine:phosphatidylserine vesicles. These studies suggest that neutral phospholipids lead to exposure of a site, probably on thrombomodulin, capable of recognizing the Gla domain of protein C.     

Functionally active thrombomodulin is present in human platelets

We found functionally active thrombomodulin in human platelets (60 +/- 18 molecules per platelet). Protein C appeared not to be activated by thrombin with gel-filtered platelets. However, the activation of protein C by thrombin was accelerated by thrombin-stimulated and washed platelets. This cofactor activity of the platelets was neutralized by the anti-lung thrombomodulin-F(ab')2. From the Triton X-extract of platelets, thrombomodulin was partially purified by diisopropylphosphoryl-thrombin-agarose affinity chromatography. The Mr of the predominant platelet thrombomodulin was 78,000 before and 109,000 after reduction on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, values identical to those of placental thrombomodulin. The specific activity of the cofactor activity, apparent Kd (0.4 nM) for thrombin and Km (0.67 microM) for protein C of platelet thrombomodulin were also identical to those of placenta thrombomodulin. Thrombomodulin may play a role in activation of protein C on the surface of platelets.     

A 10-kDa cyanogen bromide fragment from the epidermal growth factor homology domain of rabbit thrombomodulin contains the primary thrombin binding site

We have isolated a fragment (approximately equal to 10 kDa) of thrombomodulin containing the fifth and sixth epidermal growth factor (EGF)-like regions which retains thrombin binding capacity. The amino-terminal sequence of a 50-kDa active fragment of thrombomodulin derived from elastase proteolysis begins 11 residues before the first EGF-like structure of native thrombomodulin. Subsequent digestion with cyanogen bromide yields a 10-kDa thrombin binding fragment. The amino-terminal sequence of this fragment starts at the fifth EGF-like structure (Phe407). The amino acid composition suggests that this fragment contains the fifth and sixth EGF-like structures with a total of approximately 77 residues. This fragment lacks cofactor activity, but acts as a competitive inhibitor for protein C activation (Ki = 8.6 +/- 1.4 nM). We propose that the fifth and sixth EGF-like structures contain the thrombin binding site of thrombomodulin.     

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.     

Further localization of binding sites for thrombin and protein C in human thrombomodulin

To elucidate the binding sites for thrombin and protein C in the six epidermal growth factor (EGF) domains of human thrombomodulin, recombinant mutant proteins were expressed in COS-1 cells. Mutant protein EGF456, which contains the fourth, fifth, and sixth EGF domains from the NH2 terminus of thrombomodulin, showed complete cofactor activity in thrombin-catalyzed protein C activation, as did intact thrombomodulin or elastase-digested thrombomodulin. EGF56, containing the fifth and sixth EGF domains, did not have cofactor activity; but EGF45, containing the fourth and fifth EGF domains, had about one-tenth of the cofactor activity of EGF456. Thrombin binding to attached recombinant thrombomodulin (D123) was inhibited by EGF45 as well as by EGF56. A synthetic peptide (ECPEGYILDDGFICTDIDE), corresponding to Glu-408 to Glu-426 in the fifth EGF domain, inhibited thrombin binding to attached thrombomodulin (D123) with an apparent Ki of 95 microM. At Ca2+ concentrations of 0.25-0.3 mM, intact protein C was maximally activated by thrombin in the presence of EGF45, EGF456, or EGF1-6, which contains the first to sixth EGF domains; but such maximum cofactor activity was not observed when gamma-carboxyglutamic acid-domainless protein C was used. These findings suggest that: 1) thrombin binds to the latter half of the fifth EGF domain; and 2) protein C binds to the fourth EGF domain of thrombomodulin through Ca2+ ions.     

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.     

Ca2+-calmodulin-dependent phosphorylation and passive transport of Ca2+ in the myocardial sarcolemma

Highly purified vesicles of rabbit myocardium sarcolemma with predominant inside-out orientation possess the Ca2+-calmodulin-dependent protein kinase activity. At optimal concentrations of calmodulin (0.5 microM) and Ca2+ (0.1 mM), the activity of protein kinase is 0.21 nmol 32P X min X mg of protein. The Km(app) value for ATP is 3.0 X 10(-6) M, V = 0.27 nmol 32P X mg of protein X min. Endogenous Ca2+-calmodulin-dependent protein kinase phosphorylates four protein substrates in sarcolemmal vesicles (Mr = 145, 22, 11.5, and 6-8 KD). Studies with passive efflux of Ca2+ from the SL vesicles showed that the Ca2+-calmodulin-dependent phosphorylation of protein components of sarcolemma inhibits this reaction.     

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.     

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.     

The effect of phospholipids on the activation of protein C by the human thrombin-thrombomodulin complex.

Human thrombomodulin, an endothelial-cell-membrane glycoprotein, has been purified from placenta by Triton X-100 extraction and by affinity chromatography on concanavalin A-Sepharose and thrombin-Sepharose. It has been characterized by its ability to promote the activation of human protein C by human alpha-thrombin in the presence of Ca2+ and fulfilled the requirements of a cofactor. Reconstitution of thrombomodulin into phospholipid vesicles containing anionic phospholipids resulted in an increased rate of activation of protein C. Cardiolipin and vesicles containing phosphatidylcholine/phosphatidylserine (1:1, w/w) were the most effective. The apparent Km of the thrombin-thrombomodulin complex for protein C was 2 microM. It was not changed in the presence of phospholipid, whereas the Vmax. could be apparently increased up to 3.2-fold depending on the phospholipid and on its concentration, the catalytic-centre activity reaching 15.7 mol of activated protein C formed/min per mol of thrombin. Above their optimal concentrations, phospholipids inhibited the amidolytic activity of activated protein C. Phospholipids had no effect on the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues. These results show that the positive effect of anionic phospholipids in the activation of protein C by the thrombin-thrombomodulin complex involves a Ca2+-dependent interaction between protein C and phospholipids. They suggest that the enhancement of thrombomodulin activity by such phospholipids may be of functional significance.     

Substructure of skeletal myosin subfragment 1. Preferential destabilization of a domain by methanol and its effect on catalytic activity

Evidence is presented that in increasing concentrations of methanol the structure of the subfragment 1 is perturbed in such a way that the Mr = 50,000 central portion of the associated heavy chain is preferentially unfolded. This unfolding is accompanied by the loss in ATPase function where the rate of inactivation can be correlated with the loss in the amount of the Mr = 50,000 fragment generated under standard tryptic digestion conditions. The residual protein appears to be a soluble aggregate of a complex of the Mr = 27,000, 21,000, and light chain with no intact Mr = 50,000 fragment. Tryptic digestions in the presence of MgATP are restricted to the usual linker regions and the Mr = 50,000 fragment is completely protected from attack. Binding of actin to subfragment 1 also results in the protection of the Mr = 50,000 segment and of the Mr = 50,000/21,000 junction from tryptic attack. The data suggest that, in terms of methanolic perturbation, the subfragment 1 appears to be comprised of two domains with differential stability. One domain appears to be the central Mr = 50,000 segment of the heavy chain which is preferentially unfolded by methanol and requires the presence of MgATP or of actin for stabilization. The other domain is more stable and appears to consist of the interacting Mr = 27,000, 21,000, and light chain. The results also suggest that the integrity of the Mr = 50,000 segment is essential for the ATPase function of the protein.     

Two latent metalloproteases of human articular cartilage that digest proteoglycan

Human articular cartilage contains very low levels of metalloprotease activity; the activity in 1 g of cartilage is approximately equivalent to the activity of 1 microgram of trypsin. Development of a sensitive assay, based on the digestion of radioactive proteoglycan, has made it possible to study protease activity in 1-2-g specimens of cartilage. Cartilage was extracted with Tris buffer in the cold and with Tris buffer containing 10 mM CaCl2 at 60 degrees C. The extracts were passed through Sepharose 6B; two major and two minor metalloprotease activities were detected. A neutral metalloprotease activity, pH optimum 7.4, was found as a latent form of Mr = 56,000. It could be activated with aminophenylmercuric acetate or trypsin with a resultant decrease of Mr to 40,000. An acid metalloprotease, pH optimum 5.3, also occurred as a latent form of Mr = 50,000. Activation converted this to Mr = 35,000. Removal of calcium ions by dialysis reduced the activity of the neutral enzyme by 80-85% and of the acid enzyme by 100%. Both activities were restored by 10 mM Ca2+. Both enzymes were completely inhibited by 1 mM o-phenanthroline in the presence of excess calcium. This inhibition was overcome by 1 mM Zn2+ and, to a lesser extent, by Co2+. These proteases may be important in the metabolism of the cartilage matrix and in its destruction in osteoarthritis.     

Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs.

Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux.     

Mechanism of passive Ca2+ permeability of vesicular sarcolemmal preparations from rat hearts

Vesicular sarcolemmal preparations isolated from rat hearts were characterized by high total ATPase (4.32 +/- 0.57 mumol/min per mg), adenylate cyclase (121 +/- 11 pmol/min per mg) and creatine kinase (1.73 +/- 0.35 mumol/min per mg) activities as well as Na-Ca exchange specific to sodium. ATPase activity was inhibited with digitoxigenin by 50-70% and was not changed by ouabain, ionophore A23187 or oligomycin. Sarcolemmal vesicles bound [3H]digitoxigenin and [3H]ouabain in isotonic medium in the presence of Pi and Mg2+. The number of binding sites for hydrophobic digitoxigenin (N = 237 pmol/mg) was several-times higher than that for hydrophilic ouabain (N = 32.7 pmol/mg). These data show that sarcolemmal preparations were not significantly contaminated by mitochondria and sarcoplasmic reticulum and consisted mostly of inside-out vesicles. Incubation of these vesicles with 45Ca2+ (0.5-10 mM) led to penetration of the latter into the vesicles with the following binding characteristics: number of binding sites (N = 20.5 +/- 4.6 nmol/mg, Kd approximately equal to 2.0 mM). Ca2+ binding to the inner surface of vesicles was proved by the following facts: (1) Ca2+ ionophore A23187 increased slightly total intravesicular Ca2+ content but markedly accelerated Ca2+ efflux along its concentration gradient; (2) gramicidin and osmotic shock showed a similar accelerating effect. Ca2+ efflux from the vesicles along its concentration gradient ([Ca2+]i/[Ca2+]e = 2.0 mM/0.1 microM) was inhibited by Mn2+, Co2+, and verapamil when they acted inside the vesicles. The rate of Ca2+ efflux was hyperbolically dependent on intravesicular Ca2+ concentration (Km approximately equal to 2.9 mM). These data reveal that Ca2+ efflux from sarcolemmal vesicles is controlled by Ca2+ binding to the sarcolemmal membrane. Ca2+ efflux from the vesicles was stimulated 1.7--times after incubation of vesicles with 0.2 mM MgATP or MgADP and 15-times after treatment with 0.2 mM adenylyl beta, gamma-imidodiphosphate. Enhancement in the rate of Ca2+ efflux correlated with the increase in the intravesicular Ca2+ content. ATP-stimulated Ca2+ efflux was suppressed by verapamil and was nonmonotonically dependent upon the transmembrane potential created by the K+ concentration gradient in the presence of valinomycin, Ca2+ efflux being slower at extreme values of membrane potential (+/- 80 mV).     

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.     

Monoclonal antibodies for human thrombomodulin which recognize binding sites for thrombin and protein C

Monoclonal antibodies for human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C, were prepared and their epitopes characterized. All six antibodies (MFTM-1-MFTM-6) bound to an elastase-digested active fragment of thrombomodulin, which contains six consecutive EGF domains. Binding of thrombomodulin to these antibodies did not depend on Ca2+ concentration. MFTM-4, MFTM-5, and MFTM-6 strongly inhibited protein C activation by thrombin and thrombomodulin. MFTM-4 and MFTM-5 inhibited thrombin binding to fixed thrombomodulin and bound to a recombinant mutant EGF456 protein, which contained the fourth, fifth, and sixth EGF domains of thrombomodulin. However, MFTM-6 did not inhibit thrombin binding to thrombomodulin and did not bind to EGF456 protein. Binding of thrombomodulin to fixed MFTM-4 or MFTM-5 was competitively inhibited by a recombinant mutant EGF45 protein which contained the fifth and sixth EGF-domains. These results suggest that epitopes of MFTM-4 and MFTM-5 are located in the fifth EGF domain of thrombomodulin. Thus, the binding site for thrombin is located in the fifth EGF domain. These results also suggest that an epitope for MFTM-6 is located at a region near the binding site for gamma-carboxyglutamic acid residues of protein C via Ca2+ on thrombomodulin.     

Characterization of a soluble Mr-30,000 catalytic fragment of the neuronal calmodulin-dependent protein kinase II

Chymotryptic digestion of postsynaptic densities releases a soluble, catalytically active fragment of the alpha (Mr 50,000) subunit of the neuronal cytoskeletal calmodulin-dependent protein kinase II. The purified soluble form of the kinase likewise yields the fragment. Denaturation of the enzyme results in more extensive proteolytic degradation. 125I-Iodopeptide maps of the isolated catalytic portions of both forms of the enzyme are similar and are contained within the map of the isolated alpha subunit. Catalytic fragments of both forms of the enzyme comigrate on two-dimensional SDS-PAGE/isoelectric focusing with pI 6.7-7.2. The fragment phosphorylates microtubule-associated protein (MAP-2) but is not activated by Ca+2/calmodulin nor is it inhibited by trifluoperazine. Km values for MAP-2 and ATP are indistinguishable from those of the holoenzyme, while the Vmax is similar to that of the holoenzyme activated with Ca+2/calmodulin. Overlays of Western blots of fragment with 125I-calmodulin shows a loss of calmodulin binding. Both the number of phosphorylation sites and the ability to autophosphorylate are markedly reduced in the catalytic fragment. Evaluation of the hydrodynamic parameters of the purified fragment yielded Mr value of 25,600 with a frictional ratio (f/f0) of 1.12; the Mr value determined by SDS-PAGE was 30,000. Thus, the catalytic fragment appears to represent an activated form of the kinase with a monomeric, globular structure unlike the native enzyme which exhibits oligomerization and cytoskeletal association. These results are consistent with a tertiary structure for the calmodulin-dependent protein kinase that contains distinct domains responsible for catalytic activity, regulation by calmodulin, cytoskeletal association and the multimeric organization of enzyme subunits.     

Limited proteolytic digestion of coated vesicle assembly polypeptides abolishes reassembly activity

We have previously identified a fraction containing several assembly polypeptides (AP) that promotes reassembly of clathrin into vesicle-free coat structures [Zaremba S, Keen JH: J Cell Biol 97:1339, 1983]. The AP are prepared from purified bovine brain-coated vesicles by extraction with 0.5 M TRIS-HCl followed by Sepharose CL-4B column chromatography. Centrifugation in sucrose gradients under nonassembly conditions supports earlier observations suggesting that four active polypeptides in the AP preparation, of Mr approximately 110,000, 100,000, 50,000, and 16,500 are present in a discrete complex that is incorporated as a unit into reassembled coats. The 16,500-dalton polypeptide does not coelectrophorese with authentic bovine brain calmodulin and does not exhibit calmodulin's Ca2+-induced shift in electrophoretic mobility. When the partially purified AP fraction was digested with elastase, the Mr approximately 110,000 and 100,000 polypeptides were rapidly degraded with little or no effect on the Mr approximately 50,000 and 16,500 bands. This treatment abolished the in vitro coat-forming ability of the AP fraction and the loss of activity closely parallels the loss of the Mr approximately 100,000 band. Disappearance of the Mr approximately 110,000 and 100,000 bands is accompanied by the generation of new bands at Mr approximately 76,000 and 65,000. When the elastase-treated AP is examined by sucrose gradient sedimentation in nonassembly buffers, the new bands continue to cosediment with the Mr approximately 50,000 and 16,500 polypeptides. This indicates that the elastase digestion has cleaved off a fragment of the Mr approximately 110,000 and 100,000 bands, leaving behind a truncated, inactive AP complex. A protein kinase activity has been detected in coated vesicle preparations that utilizes the 50,000-dalton AP as its preferred substrate [Keen JH, Zaremba S: J Cell Biol 97:174a, 1983]. Elastase treatment does not abolish this activity, indicating that the kinase by itself is not sufficient for maintaining reassembly activity.     

Interaction of clotting factor V heavy chain with prothrombin and prethrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation

Changes in the affinity of the heavy subunit of blood coagulation factor Va (Vh) for prothrombin are thought to be important in regulating the rate of thrombin production. Using analytical ultracentrifugation, we have measured the affinity of bovine Vh for prothrombin and for the prethrombin 1 fragment of prothrombin at 23.3 degrees C, pH 7.65, in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 0.1 mM benzamidine, and either 2 mM Ca2+ or 2 mM ethylenediaminetetraacetate (EDTA). Under these conditions a 1:1 complex of Vh with prothrombin is formed that is governed by a dissociation constant (Kd) of 10 microM, regardless of whether the buffer contains Ca2+ or EDTA. An identical Kd is observed when prethrombin 1 is substituted for prothrombin. This indicates that the fragment 1 portion of prothrombin, containing the gamma-carboxyglutamic acid residues, does not influence the association. Substitution of human prethrombin 1 for the bovine molecule also results in a 1:1 Vh-prethrombin 1 complex governed by a slightly weaker Kd (27 microM). Discrete proteolysis of bovine Vh by the anticoagulant activated protein C converts the Vh to a form with little or no affinity for prethrombin 1 (Kd greater than 1 mM), without detectable change in the mass of the Vh.