Evolutionary conservation of the allosteric activation of factor VIIa by tissue factor in lamprey.

Essentials Tissue factor (TF) enhances factor VIIa (FVIIa) activity through structural and dynamic changes. We analyzed conservation of TF-activated FVIIa allosteric networks in extant vertebrate lamprey. Lamprey Tf/FVIIa molecular dynamics show conserved Tf-induced structural/dynamic FVIIa changes. Lamprey Tf activation of FVIIa allosteric networks follows molecular pathways similar to human. SUMMARY: Background Previous studies have provided insight into the molecular basis of human tissue factor (TF) activation of activated factor VII (FVIIa). TF-induced allosteric networks of FVIIa activation have been rationalized through analysis of the dynamic changes and residue connectivities in the human soluble TF (sTF)/FVIIa complex structure during molecular dynamics (MD) simulation. Evolutionary conservation of the molecular mechanisms for TF-induced allosteric FVIIa activation between humans and extant vertebrate jawless fish (lampreys), where blood coagulation emerged more than 500 million years ago, is unknown and of considerable interest. Objective To model the sTf/FVIIa complex from cloned Petromyzon marinus lamprey sequences, and with comparisons to human sTF/FVlla investigate conservation of allosteric mechanisms of FVIIa activity enhancement by soluble TF using MD simulations. Methods Full-length cDNAs of lamprey tf and f7 were cloned and characterized. Comparative models of lamprey sTf/FVIIa complex and free FVIIa were determined based on constructed human sTF/FVIIa complex and free FVIIa models, used in full-atomic MD simulations, and characterized using dynamic network analysis approaches. Results Allosteric paths of correlated motion from Tf contact points in lamprey sTf/FVIIa to the FVIIa active site were determined and quantified, and were found to encompass residue-residue interactions along significantly similar paths compared with human. Conclusions Despite low conservation of residues between lamprey and human proteins, 30% TF and 39% FVII, the structural and protein dynamic effects of TF activation of FVIIa appear conserved and, moreover, present in extant vertebrate proteins from 500 million years ago when TF/FVIIa-initiated extrinsic pathway blood coagulation emerged.

6-O-sulfotransferase-1 represents a critical enzyme in the anticoagulant heparan sulfate biosynthetic pathway.

Using recombinant retroviral transduction, we have introduced the heparin/heparan sulfate (HS) 3-O-sulfotransferase 1 (3-OST-1) gene into Chinese hamster ovary (CHO) cells. Expression of 3-OST-1 confers upon CHO cells the ability to produce anticoagulantly active HS (HS(act)). To understand how 6-OST and other proteins regulate HS(act) biosynthesis, a CHO cell clone with three copies of 3-OST-1 was chemically mutagenized. Resulting mutants that make HS but are defective in generating HS(act) were single-cell-cloned. One cell mutant makes fewer 6-O-sulfated residues. Modification of HS chains from the mutant with pure 6-OST-1 and 3'-phosphoadenosine 5'-phosphosulfate increased HS(act) from 7% to 51%. Transfection of this mutant with 6-OST-1 created a CHO cell line that makes HS, 50% of which is HS(act). We discovered in this study that (i) 6-OST-1 is a limiting enzyme in the HS(act) biosynthetic pathway in vivo when the limiting nature of 3-OST-1 is removed; (ii) HS chains from the mutant cells serve as an excellent substrate for demonstrating that 6-OST-1 is the limiting factor for HS(act) generation in vitro; (iii) in contradiction to the literature, 6-OST-1 can add 6-O-sulfate to GlcNAc residues, especially the critical 6-O-sulfate in the antithrombin binding motif; (iv) both 3-O- and 6-O-sulfation can be the final step in HS(act) biosynthesis in contrast to prior publications that concluded 3-O-sulfation is the final step in HS(act) biosynthesis; (v), in the presence of HS interacting protein peptide, 3-O-sulfate-containing sugars can be degraded into disaccharides by heparitinase digestion as demonstrated by capillary high performance liquid chromatography coupled with mass spectrometry.

Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation.

CD39, or vascular adenosine triphosphate diphosphohydrolase, has been considered an important inhibitor of platelet activation. Unexpectedly, cd39-deficient mice had prolonged bleeding times with minimally perturbed coagulation parameters. Platelet interactions with injured mesenteric vasculature were considerably reduced in vivo and purified mutant platelets failed to aggregate to standard agonists in vitro. This platelet hypofunction was reversible and associated with purinergic type P2Y1 receptor desensitization. In keeping with deficient vascular protective mechanisms, fibrin deposition was found at multiple organ sites in cd39-deficient mice and in transplanted cardiac grafts. Our data indicate a dual role for adenosine triphosphate diphosphohydrolase in modulating hemostasis and thrombotic reactions.

A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state.

The activity of the coagulation system is regulated, in part, by the interaction of thrombin with the endothelial cell receptor thrombomodulin with subsequent generation of activated protein C and suppression of thrombin production. Our previous investigation demonstrated that ablation of the thrombomodulin gene in mice causes embryonic lethality before the assembly of a functional cardiovascular system, indicating a critical role for the receptor in early development. In the current study, we show that a single amino acid substitution in thrombomodulin dissociates the developmental function of the receptor from its role as a regulator of blood coagulation. Homozygous mutant mice with severely reduced capacity to generate activated protein C or inhibit thrombin develop to term, and possess normal reproductive performance. The above animals exhibit increased fibrin deposition in selected organs, which implies tissue specific regulation of the coagulation system that is supported by further evidence from the examination of mice with defects in fibrinolysis. The thrombomodulin-deficient animals provide a murine model to examine known or identify unknown genetic and environmental factors that lead to the development of thrombosis.

The purification of megapoietin: a physiological regulator of megakaryocyte growth and platelet production.

The circulating blood platelet is produced by the bone marrow megakaryocyte. In response to a decrease in the platelet count, megakaryocytes increase in number and ploidy. Although this feedback loop has long been thought to be mediated by a circulating hematopoietic factor, no such factor has been purified. Using a model of thrombocytopenia in sheep, we have identified an active substance called megapoietin, which stimulated an increase in the number and ploidy of megakaryocytes in bone marrow culture. Circulating levels of this factor could be quantified with this assay and were found to be inversely proportional to the platelet count of the sheep. Levels increased from < 0.26 pM in normal sheep to 25-40 pM in thrombocytopenic sheep. From large amounts of thrombocytopenic sheep plasma we have purified a 31,200-Da protein and found that it retained the ability to stimulate both megakaryocyte number and ploidy in vitro. Injection of partially purified megapoietin into rats stimulated a 24% increase in megakaryocyte number and a 60% increase in mean ploidy as well as a 77% increase in the platelet count. Sheep platelets bound megapoietin and the amount of platelets required to eliminate half the activity in vitro was close to the amount associated with this same level of activity in vivo. We believe that megapoietin is the physiologically relevant mediator of megakaryocyte growth and platelet production. Moreover, our data suggest that the level of megapoietin is directly determined by the ability of platelets to remove megapoietin from the circulation.

Selection of an HEL-derived cell line expressing high levels of platelet factor 4.

A platelet factor 4 (PF4)-expressing cell line, HELNeo, was derived from the human erythroleukemia cell line, HEL. This was achieved by stable transfection of HEL cells with a construct containing the rat PF4 promoter driving the gene coding for resistance to neomycin, followed by selection of neomycin-resistant clones. HELNeo cells were all nonadhering and about 5% of the cells had polyploid nuclei (> or = 8N), as compared with 1% in HEL cells. Immunohistochemistry showed that about 90% of the HELNeo cells contained PF4, whereas only approximately 5% of the HEL cells contained PF4. No significant parallel enrichment was observed for other megakaryocytic markers, such as the glycoprotein complex IIb/IIIa, von Willebrand factor, and platelet activation-dependent granule to external membrane glycoprotein (PADGEM), which were present to a similar extent in both HEL and HELNeo lines. The increased expression of PF4 in HELNeo cells was confirmed by transient expression assays and was associated with a fivefold increase in trans-acting factors binding to the PF4 promoter. These cells should be a rich source for purifying trans-acting factors binding to the PF4 gene. Moreover, our study shows how a lineage-specific promoter may be used to generate lineage-specific cell lines from a multilineage hematopoietic cell line.

Transcriptional regulation of the thrombomodulin gene.

The transcriptional start sites of the endogenous human thrombomodulin (TM) gene and transiently expressed TM promoter/CAT gene constructs were defined by nuclease S1 mapping which showed two closely spaced sites at +1 and +6, respectively. Transient expression and in vitro transcription assays of 5' and internal deletion mutants of the TM promoter/CAT gene constructs reveal that the region from -72 to -29 exhibits a positive acting domain which is essential for transcriptional activity, whereas the region from -373 to -225 possesses two positive acting subdomains, -343 to -277 and -245 to -225, which together augment transcriptional activity by about 40%. Electrophoretic mobility shift assays with a duplex oligonucleotide corresponding to -72 to -29 and DNase I footprinting experiments show two specific interaction products which individually or cooperatively protect the DNA sequence from about -60 to -30. These components are essential for TM gene transcription since affinity fractionation of nuclear extracts with a duplex oligonucleotide corresponding to -72 to -29 depletes the above interaction products and specifically inhibits in vitro transcription activity of the promoter, whereas addition of the eluted components specifically restores in vitro transcription activity of the promoter. Electrophoretic mobility shift assays with duplex oligonucleotides corresponding to -294 to -215, as well as -373 to -295 and DNase I footprinting experiments show two specific interaction products which individually bind to the two subdomains but not -72 to -29 and protect the coding and noncoding strands from -245 to -225, and the noncoding strand from -337 to -314, respectively. Transient expression studies reveal that the TM promoter construct starting at -51 and including the TATA box is responsive to TNF only in cell lines exhibiting sensitivity of the endogenous receptor gene to cytokine, whereas other promoter constructs possessing a TATA box sequence are insensitive to TNF in all cell types. Based upon the above data, the regulatory events involved in TNF-dependent transcriptional regulation of the TM gene can be defined with the experimental tools and conceptual framework developed by the present investigation.

Transcriptional regulation of the rat platelet factor 4 gene: interaction between an enhancer/silencer domain and the GATA site.

We used various segments of the 5' upstream region of the rat platelet factor 4 (PF4) gene coupled to the human growth hormone gene and heterologous promoters to identify domains which are critical for tissue-specific expression. Transient expression experiments with rat bone marrow cells and other cell lines revealed a complex interplay between a core promoter domain from -97 to the transcriptional start site and an enhancer/silencer domain from -448 to -112. The core promoter contains a GATA site at -31 to -28 whose mutation to TATA or AATA decreases tissue specificity and moderately affects expression in megakaryocytes as well as a positively acting subdomain from -97 to -83 whose removal decreases overall transcription without affecting tissue specificity. The enhancer/silencer domain possesses three positively acting subdomains from -380 to -362, -270 to -257, and -137 to -120 as well as a negatively acting subdomain at -184 to -151 which is able to reduce overall transcription but has no effect on tissue specificity. The subdomain from -380 to -362 is most critical in restricting gene expression driven either by the PF4 promoter or by a heterologous promoter to the megakaryocytic lineage. The subdomains from -270 to -257 and -137 to -120 function together with the subdomain from -380 to -362 to somewhat increase tissue specificity. Simultaneous mutation of the GATA site and deletion of either the whole enhancer/silencer domain or the subdomain from -380 to -362 or -137 to -120 reduce transcription in megakaryocytes by 10- to 30-fold. On the basis of the above-described results, we propose that the megakaryocyte-specific enhancer/silencer domain and the GATA site are responsible for high-level expression of the PF4 gene in a lineage-specific manner.

Selective targeting of gene products with the megakaryocyte platelet factor 4 promoter.

We have used the 1.1 kilobases of the 5' upstream region of the platelet factor four (PF4) gene coupled to the prokaryotic beta-galactosidase gene to generate two lines of transgenic mice that express this construct. Studies of blood, bone marrow, spleen, and thymus reveal that platelets are the only circulating blood cells and megakaryocytes are the only hematopoietic precursor cells that possess the prokaryotic enzyme. The lack of transgene expression in brain, heart, intestine, kidney, liver, lung, and skeletal muscle was established by in situ staining of tissue sections as well as kinetic assay of tissue homogenates. These data suggest that this domain of the PF4 promoter contains most, if not all, of the tissue-specific region of the gene. Unexpectedly, the adrenal gland exhibits approximately 2% of the levels of beta-galactosidase possessed by megakaryocytes and the distribution of the prokaryotic enzyme corresponds to the location of mineralocorticoid-secreting cells. This result implies that either the PF4 gene is transcribed at low levels in specialized adrenal cells or that these specialized endocrine cells possess trans-acting factors similar to those that control the megakaryocyte promoter. The selective high-level expression of transgenes linked to the PF4 promoter should allow us to augment or suppress the in vivo levels of critical components in megakaryocytes and platelets and subsequently ascertain the effects of these modifications.

Human thrombomodulin gene is intron depleted: nucleic acid sequences of the cDNA and gene predict protein structure and suggest sites of regulatory control.

We have isolated a human thrombomodulin cDNA, and a human genomic clone containing the putative promoter domain, as well as the translated and untranslated regions of the endothelial cell receptor. The nucleotide sequence of the thrombomodulin cDNA allows us to provide a complete picture of the structure of this endothelial cell receptor, and to confirm its homology to the human low density lipoprotein receptor. The nucleotide sequence of the thrombomodulin gene suggests areas within the putative promoter domain that may be critical for regulating expression of the human endothelial cell receptor, indicates a potential signal peptide, and shows that no introns are present within the coding region. The overall organization of the human thrombomodulin gene is surprising because it represents an example of a gene that contains epidermal growth factor type B repeats and a membrane spanning region, which are not isolated within discrete exons.

Characterization of a thrombomodulin cDNA reveals structural similarity to the low density lipoprotein receptor.

We have isolated a partial-length cDNA for bovine thrombomodulin from a lambda gt11 bovine adrenal capillary endothelial cell expression library. This was accomplished by immunoscreening with rabbit anti-thrombomodulin IgG heteroantibody and then rescreening with the initial positive recombinant insert. The cDNA obtained was authenticated by showing that it coded for the primary structure of two separate regions of bovine thrombomodulin. The nucleotide sequence of the largest cDNA allowed us to establish the structure of about 80% of the mature thrombomodulin transcript, which encodes the C-terminal half of the polypeptide. This membrane component is structurally similar to coated-pit receptors and is organized into domains that resemble those of the low density lipoprotein receptor.

Interaction of factor XIa and antithrombin in the presence and absence of heparin.

We have studied the interaction between purified human factor XIa and antithrombin in the presence and absence of well-characterized preparations of heparin. The concentrations of hemostatic enzyme, protease inhibitor, and mucopolysaccharide were 5.76 X 10(-8) mol/L, 5.76 X 10(6) mol/L, and either 5.88 X 10(6) mol/L or 0, respectively. Kinetic investigation of this process using a tritiated factor IX substrate demonstrated that the pseudo first-order rate constants of this reaction in the presence and absence of heparin are approximately 1.0 min-1 and approximately 0.025 min-1, respectively. Thus, the rate of hemostatic enzyme-protease inhibitor complex formation is accelerated by about 40-fold in the presence of saturating levels of the mucopolysaccharide. These results were confirmed in a qualitative manner by directly monitoring the generation of factor XIa-antithrombin interaction product with sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using an antibody population specific for the protease inhibitor.

Detection of protein C activation in humans.

We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The protein C activation peptide (PCP) was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used together with a 125I-labeled tyrosinated ligand and various concentrations of unlabeled PCP to construct a double antibody RIA capable of measuring as little as 10 pM of this component. We have established that the synthetic dodecapeptide has the same immunoreactivity as the native peptide and that the reactivity of protein C is less than 1/2,000 that of PCP on a molar basis. The extremely low levels of peptide in normal individuals as well as the nonspecific contributions of plasma constituents to the immunoreactive signal, necessitated the development of a procedure by which the PCP could be reproducibly extracted from plasma and concentrated approximately 20-fold. This methodology permitted us to demonstrate that the plasma PCP levels in 17 normal donors averaged 6.47 pM, and that elevations up to 180 pM were observed in individuals with evidence of disseminated intravascular coagulation. The validity of these measurements of protein C activation is supported by the fact that, in both of these situations, the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native dodecapeptide. We have also noted that the mean PCP concentration in seven patients fully anticoagulated with warfarin averaged 2.61 pM. Our studies also show that PCP is cleared from the plasma of primates with a t1/2 of approximately 5 min. Given that the t1/2 of activated protein C is estimated to be 10-15 min, the latter enzyme appears to exert its effects on the activated cofactors of the coagulation system at concentrations considerably less than 1.0 nM.

Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells.

Previous work from our laboratory has demonstrated that both anticoagulant and nonanticoagulant heparin species can inhibit the proliferation of vascular smooth muscle cells in vivo and in vitro. In this communication, we report studies on the structure-function relationships of heparin to its antiproliferative effect on vascular smooth muscle cells. These structure-function studies were carried out by preparing discrete sizes of heparin fragments and by chemically modifying heparin. The compounds were tested for their ability to inhibit rat and calf aortic smooth muscle cell growth. The minimum fragment size which retains some growth inhibitory activity is a hexasaccharide; maximal antiproliferative activity was obtained with dodecasaccharide and larger fragments. Both O-sulfation and N-substitution were found to be important for the growth inhibitory effect. Comparison of the antiproliferative and anticoagulant activities of the different heparin species has allowed us to identify several heparin molecules which have lost their anticoagulant properties, but retain antiproliferative activity.

Purification and properties of human platelet heparitinase.

An endoglycosidase which cleaves heparin and heparan sulfate was isolated from outdated human platelets by freeze-thaw solubilization, heparin-Sepharose chromatography, DEAE-cellulose chromatography, hydroxylapatite chromatography, octyl-agarose chromatography, concanavalin A-Sepharose chromatography, and Sephacryl S-200 gel filtration. The overall extent of purification of the platelet heparitinase is about 240,000-fold and the overall yield of the enzyme is about 5.6% as compared to the initial freeze-thaw solubilization preparation. The final product is physically homogeneous as judged by disc gel electrophoresis at acidic pH as well as gel filtration chromatography and exhibits an apparent molecular weight of approximately 134,000. Furthermore, our results indicate that the above enzyme is present within platelet lysosomes. The biologic potency of the endoglycosidase was examined as a function of pH. The data show that the platelet heparitinase is maximally active from pH 5.5 to pH 7.5. However, the enzyme possesses minimal ability to cleave heparin at pH less than 4.0 or greater than 9.0. The substrate specificity of the platelet endoglycosidase was determined by identifying susceptible linkages within the heparin molecule that can be cleaved by the above component. Our studies indicate that this enzyme is only able to hydrolyze glucuronsylglucosamine linkages. Furthermore, investigation of the structure of the disaccharide which lies on the nonreducing end of the cleaved glucuronic acid residue suggests that N-sulfation of the glucosamine moiety or ester sulfation of the adjacent iduronic acid groups are not essential for bond scission.

Multiple functional domains of the heparin molecule.

Affinity-fractionated porcine heparin was randomly scissioned by chemical techniques to give hexasaccharides, octasaccharides, decasaccharides, and mucopolysaccharide fragments of approximately 14 residues and approximately 16 residues that were able to complex with the protease inhibitor. Direct measurements of the kinetic behavior of the hexasaccharides, octasaccharides, and decasaccharides showed that these fractions greatly enhanced the rate of Factor Xa inactivation by antithrombin. Indeed, these species exhibited specific molar activities that ranged from 6.9% (hexaccharide) to 60.9% (decasaccharide) of that of the heparin fragment of approximately 16 residues. However, these oligosaccharides exhibited essentially no ability to accelerate thrombin-antithrombin interactions. The avidity of the hexasaccharides, octasaccharides, and decasaccharides for the protease inhibitor increased as a function of size with the respective dissociation constants ranging from 5.5 X 10(-6) M to 2.9 X 10(-7) M. These data suggest that the region of the heparin molecule needed for catalyzing Factor Xa-antithrombin interaction is intimately related to the antithrombin binding domain. The smallest complex carbohydrate fragment that accelerated the inactivation of thrombin by antithrombin had approximately 14 residues. This fraction had an avidity for the protease inhibitor of 2.8 X 10(-7) M and specific molar activities of 140 units per mumol (thrombin neutralization) and 460 units per mumol (factor Xa inactivation). The largest heparin fragment examined contained approximately 16 residues. This fraction had an avidity for antithrombin of 2.4 X 10(-7) M and specific molar activities of 500 units per mumol (thrombin neutralization) and 560 units per mumol (Factor Xa inactivation). Detailed kinetic analyses showed that these two species are able to directly activate antithrombin to the same extent with respect to thrombin inhibition. However, the larger mucopolysaccharide fragment is also capable of approximating free enzyme with protease inhibitor.

The isolation and characterization of a specific antibody population directed against the prothrombin activation fragments F2 and F1 + 2.

We have raised antisera against human prothrombin activation fragment F2 in rabbits and have chromatographed the respective immunoglobulin G fractions on prothrombin-Sepharose, Pr1-Sepharose, and F2-Sepharose immunoadsorbents. The specific antibody population obtained was utilized to construct a double antibody radioimmunoassay capable of measuring as little as 0.8 ng/ml of this component. Our studies suggest that the immunoreactive site defined by this antibody population is most probably located within the negatively charged COOH-terminal region of F2. The immunologic expression of this area is unaffected by denaturation or reduction-alkylation of F2 as well as by attachment of polypeptide to the NH2-terminal of this component. However, the presence of covalently bound polypeptide at the COOH-terminal of F2 reduces its immunologic reactivity by 300- to 400-fold. Prothrombin, Pr1, and Pr*1, which contain the F2 region as part of their covalent structure, are at least 4000 to 7000 times less immunoreactive than F2 on a molar basis. Conversion of these components to thrombin as well as activation fragments generates the theoretically predicted level of immunoreactivity. Masking of the immunoreactive site within these zymogens is due to two phenomena. Firstly, covalent attachment of polypeptides on the COOH-terminal of the F2 segment significantly depresses the reactivity of this region. Secondly, a critical S--S bridge aids in the sequenstration of the immunoreactive site. This cross-link may facilitate interactions between the COOH-terminal of the F2 segment and other regions of the zymogen.

Activation of human prothrombin by highly purified human factors V and X-a in presence of human antithrombin.

In this communication we describe the first method for isolating human Factor V. The final product contains no other coagulation components as judged by functional assays and is physically homogeneous as shown by isofocusing gel electrophoresis. In addition, we present a means for obtaining intrinsically activated human Factor X-a. This preparation is usually homogeneous as judged by isofocusing gel electrophoresis. However, on occasion, an additional minor electrophoretic species with Factor X-a activity is observed. Furthermore, we describe the use of isoelectric focusing in sucrose density gradients to free human prothrombin from contamination by coagulation factors and other components. These homogeneous human proteins are employed to examine the conversion of prothrombin to thrombin in the presence and absence of human antithrombin. The latter component is responsible for virtually all of the plasm's capacity to neutralize Factor X-a and thrombin. In the absence of antithrombin, prothrombin (67,800) is converted to the precursor P-2 (51,600) and the fragment F-a (19,500). Subsquently, P-2 is cleaved to form the precursor P-3 (37,000), and the fragment F-b (11,500). Finally, P3 IS proteolyzed to form the heavy chain T-h (29,500) and the light chain T-L (6,500) of active thrombin. In the presence of antithrombin, an additional prothrombin conversion pathway is observed in which the zymogen is directly cleaved to form P-3 and F-A + B (30,000) prior to thrombin generation. Trace amounts of free thrombin remain uninhibited by antithrombin and could bias the zymogen activation pathway. Hirudin is known to neutralized thrombin instantaneoulsly. We demonstrate that the purified leech protein also binds to P-3 and prevents thrombin formation. When hirudin is added to activation mixtures at concentrations sufficient to virtually suppress P-3 conversion to thrombin, molecular species from both activation pathways are observed. Thus two human prothrombin conversion sequences appear to be initiated by Factor X-3 and may be of physiological significance.