Urinary 11-dehydro-thromboxane B(2) and coagulation activation markers measured within 24 h of human acute ischemic stroke.

The aim of this study was to determine the extent of change in platelet and coagulation markers in the acute phase of ischemic stroke and to assess the utility of marker measurement in stroke subtype classification. Urinary 11-dehydro-thromboxane B(2) (11-dTXB2), a marker of in vivo platelet activation, and markers of coagulation activation, including prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and fibrinogen, were measured in 25 patients with ischemic stroke within 24 h of onset of symptoms. Marker levels in patients with ischemic stroke were compared with those in 19 age-matched controls who had not taken aspirin for at least 2 weeks before sampling and 25 healthy controls. Median marker levels were significantly increased in stroke over those in age-matched controls for fibrinogen (344 vs. 289 mg/dl; P=0.030), F1+2 (1.40 vs. 0.80 nmol/l; P=0.003), and TAT (6.65 vs. 2.20 microg/l; P<0.0001). Median marker levels for seven patients with cardioembolic stroke and 18 with non-cardioembolic stroke were not significantly different for any marker test. Eight patients taking aspirin at the time of the stroke had significantly lower 11-dTXB2 values than patients not taking aspirin (964 vs. 4,314 pg/mg of creatinine; P=0.007). Stroke patients not taking aspirin had significantly higher 11-dTXB2 concentration than age-matched controls (4,314 vs. 1,788 pg/mg of creatinine; P=0.006). Coagulation and platelet activation markers are increased in the acute phase of stroke regardless of the clinical mechanism. This finding suggests that the markers may not be useful for predicting clinical subtype of ischemic stroke in the acute phase.

Combined heterozygosity of factor V leiden and the G20210A prothrombin gene mutation in a patient with cerebral cortical vein thrombosis.

Cerebral venous thrombosis (CVT) is a rare type of stroke with a variety of causes. Several reports have suggested that either factor V Leiden or G20210A prothrombin gene mutation is associated with an increased risk of CVT. The genetic thrombophilias are typically associated with other predisposing factors. We report a unique case of CVT in a patient with both the factor V Leiden and the G20210A prothrombin gene mutations without other identifiable precipitating factors in a 28-year-old white male in good health. MRI and cerebral arterial angiography showed cerebral cortical venous thrombosis. This case suggests that combined heterozygous individuals may be particularly prone to spontaneous thrombosis, like CVT.

Protein-bound heparin/heparan sulfates in human adult and umbilical cord plasma.

We used thrombin times and a competitive radiometric assay to identify, quantitate and characterize endogenous heparin-like molecules in umbilical cord (n = 58) and normal adult (n = 25) plasma. Thrombin times for cord plasma (29.6+/-3.6 s) were significantly longer (p< or = 0.0005) than those for adult plasma (18. 9+/-2.3 s), suggesting increased endogenous heparins. A radiometric assay based on the displacement of (125)I-heparin from protamine-Sepharose revealed that protease-digested plasma contained heparin/heparan sulfate, and plasma that was not digested with protease appeared not to contain heparin/heparan sulfate. More heparin/heparan sulfate was identified in cord than in adult plasma (p< or =0.05), but heparinase digestion produced significantly (p< or =0.001) reduced concentrations of heparin/heparan sulfate in only 39% of the samples. The lack of heparinase sensitivity in 61% of the protease-digested samples apparently was due to low molecular weight (LMW) heparins, for control heparin fragments of 5 kD that did not extend thrombin times were also less affected by heparinase, but the same LMW heparins were detected by radiometric assay. Despite normal thrombin times in all samples, the amounts of endogenous heparin/heparan sulfate identified in protease-digested samples by radiometric assay were of sufficient concentrations to produce inordinately prolonged thrombin times when compared with the same concentrations of unfractionated heparin. Collectively, these findings suggest the presence of a plasma reservoir of endogenous heparin/heparan sulfates in normal cord and adult plasma. These endogenous heparin/heparan sulfates are bound to plasma proteins, and an as yet undetermined proportion of these bound heparin/heparans are most likely LMW molecules.

Thrombin induces thrombomodulin mRNA expression via the proteolytically activated thrombin receptor in cultured bovine smooth muscle cells.

Thrombin, an important mitogen governing smooth muscle cell proliferation, binds to cultured bovine aortic smooth muscle cells (BASMCs) via both the proteolytically activated thrombin receptor (PATR) and thrombomodulin (TM). Although TM mRNA expression and functional activity is regulated by thrombin in human endothelial cells and mouse hemangioma cells, it remains unclear in those models whether the increased TM mRNA expression observed upon thrombin stimulation is mediated through the activation of PATR or via TM occupancy. We observed in cultured BASMCs that TM mRNA is increased threefold to sixfold by either thrombin, basic fibroblast growth factor (bFGF), or platelet-derived growth factor (PDGF). The increase in TM mRNA with thrombin is time dependent (maximal at 3 hours), a consequence of increased mRNA stability, and accompanied by increases in cell surface TM functional activity. Thrombin-induced TM mRNA was reproduced by the hexameric thrombin receptor-activating peptide (TRAP6) and augmented by a TM-specific antibody. Together, these data suggest that up-regulation of TM mRNA by thrombin is mediated via the PATR. We speculate that increases in BASMC TM mRNA and activity after thrombin may contribute to the impaired thrombus formation observed after atherosclerotic vascular injury.

Mitogen crosstalk accompanying urokinase receptor expression in stimulated vascular smooth muscle cells.

Thrombin and other mitogens regulate the expression of the urokinase-type plasminogen activator receptor (uPAR) protein and mRNA levels in bovine vascular smooth muscle cells (SMC). We investigated interactions between mitogens capable of increasing uPAR mRNA levels in SMC. Up-regulation of uPAR mRNA upon thrombin and basic fibroblast growth factor (bFGF) stimulation was preceded by a 2-3-fold transient increase in bFGF mRNA within 1 h. TGF-beta1 did not result in a significant change in bFGF mRNA levels. Platelet-derived growth factor (PDGF) while substantially enhancing uPAR mRNA levels, diminished bFGF mRNA levels by 3-4-fold. Both thrombin and bFGF induced the message for bFGF-R 2-3-fold. Thrombin also provoked a 3-4-fold rise in TGF-beta1 mRNA levels within 30 min. In summary, on the mRNA level, we demonstrated both positive as well as negative feed-back mechanisms between different mitogens, among them bFGF revealing in addition to autoinduction also up-regulation of the transcript concentration of its own receptor. Thus, cooperation and possible amplification of mitogenic effects might be implicated in the fine-tuned regulation of uPAR mRNA in stimulated bovine aorta SMC.

Effect of thrombin, the thrombin receptor activation peptide, and other mitogens on vascular smooth muscle cell urokinase receptor mRNA levels.

Bovine vascular smooth muscle cells (SMC) express the urokinase-type plasminogen activator receptor (u-PAR) claimed to be important in cell invasion. Receptor numbers and affinity are regulated by thrombin and several other mitogens involved in SMC proliferation. We investigated the effects of these mitogens on u-PAR mRNA levels. On continuous thrombin stimulation the u-PAR message in SMC was 10 +/- 2.3-fold elevated reaching a maximum between 6 and 9 hours and declining to control values within 48 hours. Thrombin present for 30 minutes on the cell surface produced similar effects. Stimulation with the thrombin receptor activation peptide S-F-L-L-R-N representing the NH2-terminus of the tethered ligand also increased u-PAR mRNA levels with an identical time course. D-Phe-Pro-Arg-chloromethyl ketone (PPACK) active site blocked thrombin and the catalytically inactive thrombin mutant S205A did not affect u-PAR mRNA levels. Thrombin stimulation also resulted in a 2 +/- 0.2-fold transient increase in thrombin receptor mRNA preceding the rise in u-PAR message. Transforming growth factor beta 1 (TGF beta 1) and platelet-derived growth factor (PDGF) showed similar time courses for the elevation of u-PAR mRNA levels with a maximal 5.5 +/- 0.9 and 12 +/- 2.5-fold increase, respectively. Basic fibroblast growth factor (bFGF) and phorbol myristate acetate (PMA) showed a more prolonged effect increasing u-PAR mRNA levels 8 +/- 2.0-fold and 12.3 +/- 2.5-fold, respectively, within 6 hours but remaining 5 to 10-fold elevated at 48 hours. In order to decide if the u-PAR mRNA increase was due to message stabilization or a consequence of transcriptional activation we used the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) during the stimulation experiments. u-PAR mRNA levels on TGF beta 1 stimulation of SMC decayed after the addition of DRB indicating that enhancement of transcriptional activity was involved in the induction. In contrast, the time course of u-PAR mRNA elevation on thrombin, bFGF, and PMA stimulation was not significantly altered in the presence of DRB suggesting that in these latter cases u-PAR mRNA message accumulation was at least in part due to mRNA stabilization. Increased transcriptional activity, mRNA stabilization and expression of u-PAR protein on the SMC surface in response to growth factors may facilitate enhanced cell surface protease activity, cell migration, and development of atheromatous lesions.

Molecular cloning of cDNA for the bovine urokinase-type plasminogen activator receptor.

We isolated a full length cDNA clone for bovine u-PAR from a bovine aorta endothelial cell cDNA library and compared the structural properties of this receptor protein to the published human and murine sequences. The bovine u-PAR cDNA clone spans a nucleotide sequence stretch of 1335 bp. The open reading frame contains 330 amino acids with a 20 amino acid long putative signal peptide. The mature protein contains 6 potential N-linked glycosylation sites and a high cysteine content (9%). Bovine u-PAR revealed three homologous internal structural repeats. The NH2-terminal repeat containing the u-PA binding site showed 54% identity to the human and murine NH2-terminal domain, compared to 64% identity between human and mouse u-PAR. Southern blot analysis of genomic DNAs from 9 eukaryotic species suggests that the u-PAR gene is conserved in man, monkey, and cow.

Identification of the predominant glycosaminoglycan-attachment site in soluble recombinant human thrombomodulin: potential regulation of functionality by glycosyltransferase competition for serine474.

Thrombomodulin (TM) is an endothelial cell thrombin receptor that converts thrombin from a procoagulant to an anticoagulant enzyme. It has previously been shown that TM is expressed in both a high-M(r) form containing chondroitin sulphate and a low-M(r) form lacking this modification. Site-directed mutagenesis of a soluble human TM derivative (TMD1) was employed to determine the attachment site(s) of this functionally important oligosaccharide on the core protein. Although there are four serine residues within the Ser/Thr-rich domain of TMD1 that might support glycosaminoglycan assembly, our analysis demonstrates that the primary site of attachment is at Ser474, and evidence is presented for low levels of attachment at Ser472. It was possible to improve the overall degree of attachment by mutating Ser472 to glutamic acid (so as to conform Ser474 to the xylosyltransferase acceptor consensus acidic-Gly-Ser-Gly-acidic); however, a significant proportion (approx. 35%) of the total TM still lacked a glycosaminoglycan moiety. Mutants that possess a substitution for Ser474 show an increased mobility of their low-M(r) form on SDS/PAGE compared with native TMD1. Isolation and sequencing of a C-terminal peptide demonstrated that this serine is modified in the low-M(r) form of native TMD1. An apparent 'acceptor consensus overlap' at Ser474 suggests that the mechanism behind the glycosaminoglycan split of TM may involve a competition for substrate between xylosyltransferase and N-acetylgalactosaminyltransferase.

Recombinant human thrombomodulin attenuates human endothelial cell activation by human thrombin.

Two glycoforms of recombinant human thrombomodulin (TM; TMD1-105 and TMD1-75), an endothelial cell membrane protein, were tested for their ability to alter thrombin-induced activation of cultured human umbilical vein endothelial cells (HUVECs). After stimulation with 10 nmol/L thrombin, HUVEC generation of inositol-1,4,5-trisphosphate (IP3), a potent Ca(2+)-mobilizing second messenger, was dose-dependently blocked by TMD1-105. Both TMD1-105 (IC50 = 10 nmol/L) and TMD1-75 (IC50 = 100 nmol/L) blocked the enhanced prostacyclin synthesis by HUVEC monolayers treated with 10 nmol/L thrombin. HUVEC monolayer permeability to Evans blue dye-labeled albumin increased from 0.125 +/- 0.06 microL/min in control experiments to 0.380 +/- 0.09 microL/min after treatment with 100 nmol/L thrombin (P < .05). Incubation of HUVECs with TMD1-105 alone (600 nmol/L) had no effect (0.114 +/- 0.04 microL/min) on basal permeability. In contrast, incubation of 100 nmol/L thrombin with 600 nmol/L TMD1-105 reduced this increase in HUVEC permeability to almost control levels (0.142 +/- 0.06 microL/min). These results demonstrate that recombinant human TM, a potent in vitro anticoagulant, also functions as an antagonist of thrombin receptor-mediated HUVEC activation. In addition to its anticoagulant functions, the high-affinity endothelial cell receptor TM may play a role in modulating endothelial cell activation by thrombin.

Role of the glycosaminoglycan component of thrombomodulin in its acceleration of the inactivation of single-chain urokinase-type plasminogen activator by thrombin.

Thrombomodulin (TM), a membrane proteoglycan on endothelial cells, binds thrombin in a 1:1 complex, accelerates the protein C activation by thrombin, promotes the thrombin inactivation by antithrombin III and inhibits the procoagulant properties of thrombin. The inactivation of single-chain urokinase-type plasminogen activator (scu-PA) by thrombin is accelerated about 70-fold by TM [De Munk, Groeneveld and Rijken (1991) J. Clin. Invest. 88, 1680-1684]. The present study investigates the role of the O-linked glycosaminoglycan moiety of TM in the latter reaction. In the presence of an excess of a fully-glycosylated soluble recombinant human TM mutant (high-Mr rec-TM), 0.11 nM thrombin inactivated 50% of 4.4 nM scu-PA in 45 min at 37 degrees C. In the presence of a soluble recombinant TM mutant lacking the glycosaminoglycans (low-Mr rec-TM), 1.9 nM thrombin was needed to inactivate 50% scu-PA, as compared with 4.7 nM thrombin in the absence of TM. Using the scu-PA inactivation assay the dissociation constant for the thrombin-TM interaction was found to be 0.4 nM for high-Mr rec-TM and 14 nM for low-Mr rec-TM. Treatment of high-Mr rec-TM with chondroitinase ABC to digest the glycosaminoglycans decreased the accelerating effect to the level of low-Mr rec-TM. A similar decrease was observed after treatment of solubilized rabbit TM with chondroitinase ABC. As expected, chondroitinase ABC had no influence on the accelerating effect of low-Mr rec-TM. The free glycosaminoglycans obtained by alkaline treatment of TM or chondroitin sulphate A also accelerated the inactivation of scu-PA by thrombin, but about 1000-fold higher concentrations than with TM were needed to obtain the same acceleration. It is concluded that the major glycosaminoglycan of TM plays a pivotal role in the inactivation of scu-PA by the TM-thrombin complex, both in the formation and in the activity of the complex.

Regulation of the urokinase-type plasminogen activator receptor on vascular smooth muscle cells is under the control of thrombin and other mitogens.

The urokinase-type plasminogen activator receptor (u-PAR) was demonstrated on cultured smooth muscle cells (SMCs) of bovine aorta. Binding of 125I-urokinase-type plasminogen activator (u-PA) was concentration dependent and saturable within 45-60 minutes. A similar concentration and time dependence was found in functional plasminogen activation studies. Human two-chain high-molecular-weight u-PA and its proenzyme (pro-u-PA) bound specifically with identical affinity (Kd). Activation of pro-u-PA was strongly accelerated on binding to SMCs and occurred only in the presence of plasminogen on the cell surface. A 100-fold molar excess of unlabeled high-molecular-weight u-PA effectively blocked binding of the radiolabeled ligands; tissue-type plasminogen activator, plasminogen, low-molecular-weight u-PA, and unrelated proteins did not. 125I-u-PA binding was abolished by a monoclonal antibody against the specific u-PA sequence responsible for u-PAR binding. Binding of u-PA sharply decreased on SMC exposure to phosphatidylinositol-specific phospholipase C, confirming the glycan phospholipid cell anchorage of u-PAR. Bovine and human alpha-thrombin (240 nM) increased the binding of 125I-u-PA fivefold, translating into an increase in the number of sites per cell from about 10(5) to 5 x 10(5) without significant change in the Kd (1.29 +/- 0.39 nM). Active site blockade of thrombin by D-Phe-Pro-Arg-chloromethyl ketone resulted in the total loss of stimulatory activity, as did the use of the inactive active site thrombin mutant, S205A. Hirugen (100 microM), which blocks the anion-binding exosite of thrombin, blocked u-PAR stimulating activity. Thus, both the catalytic activity and integrity of the exosite are important for thrombin's stimulatory activity. Other SMC mitogens (epidermal growth factor, transforming growth factor-beta 1, basic fibroblast growth factor, platelet-derived growth factor, and phorbol 12-myristate 13-acetate) increased u-PAR expression on SMCs six- to 20-fold while concomitantly increasing Kd four- to 10-fold. In all cases the induction of u-PAR was dependent on de novo protein synthesis. These observations assign a possible role for thrombin and other mitogens in u-PAR regulation, thereby influencing the pericellular proteolysis that is important in SMC migration and atheromatous plaque development.

Role of plasminogen-activator inhibitor type 1 in the pathogenesis and outcome of the hemolytic uremic syndrome.

BACKGROUND: Deposition of fibrin in glomeruli and renal failure are characteristic features of the hemolytic uremic syndrome. An inhibitor of glomerular fibrinolysis has been detected in plasma from children with this disorder. In this study, we define the inhibitor and show that its plasma level is correlated with the outcome of the disease. METHODS AND RESULTS: Plasminogen-activator inhibitor type 1 (PAI-1) in plasma was measured with an assay employing a specific monoclonal antibody in 40 consecutive children hospitalized with the hemolytic uremic syndrome: 12 who recovered adequate renal function (serum creatinine, less than or equal to 2.0 mg per deciliter [177 mumol per liter]) without dialysis, 23 who recovered adequate renal function after peritoneal dialysis, and 5 who did not recover adequate renal function after undergoing dialysis. At presentation, plasma PAI-1 levels were higher in the patients with the hemolytic uremic syndrome than in nine children with other forms of acute renal failure. That the inhibitor was PAI-1 was indicated by the fact that it was a potent inhibitor of tissue plasminogen activator, was acid-resistant, and was not inhibited by denaturation (all unique traits of PAI-1) and that it was neutralized by an antibody specific for PAI-1. Multivariate discriminant-function analysis revealed that the duration of elevated PAI-1 activity was strongly correlated with the outcome of the disease (P less than 0.001). Peritoneal dialysis reduced plasma PAI-1 levels dramatically. CONCLUSIONS: Our studies suggest that PAI-1 is the circulating inhibitor of fibrinolysis in the hemolytic uremic syndrome. Normalization of plasma PAI-1 levels (e.g., by peritoneal dialysis) is correlated with improvement in renal function. However, the possibility that increased plasma levels of PAI-1 are either causes or effects of the hemolytic uremic syndrome is not unequivocally established by these studies.

Recombinant human thrombomodulin. Regulation of cofactor activity and anticoagulant function by a glycosaminoglycan side chain.

Two glycoforms of a secretable human thrombomodulin mutant [TMD1-105 and TMD1-75; Parkinson, Grinnell, Moore, Hoskins, Vlahos & Bang (1990) J. Biol. Chem. 265, 12602-12610] were expressed in human 293 cells and used to study the role of glycosylation in the functions of this endothelial-cell thrombin receptor. Carbohydrate content analysis and intrinsic labelling with [3H]glucosamine and [35S]sulphate showed that TMD1-105 contained a chondroitin sulphate whereas TMD1-75 did not. Other than chondroitin sulphate, the carbohydrate contents of the two glycoforms were identical, indicating similar glycosylation patterns at other O-linked and N-linked sites in the two glycoforms. The properties of TMD1-105 were converted into those of TMD1-75 by chondroitin ABC lyase digestion. Trypsin digestion of labelled TMD1-105 permitted isolation of two overlapping peptides that contained chondroitin sulphate, spanned the entire O-glycosylation domain and had O-glycosylation sites at Ser-492, Ser-498, Thr-500, Thr-504 and Thr-506. The chondroitin sulphate-attachment site was assigned to Ser-492 as this residue is conserved in mouse and bovine thrombomodulin and lies within a sequence Ser-Gly-Ser-492-Gly-Glu-Pro, which has strong similarity to chondroitin sulphate attachment sites in other proteoglycans. Five peptides with N-linked carbohydrate were also isolated and contained glycosylation sites in the lectin-like domain (Asn-47, Asn-115, Asn-116) and in the fourth (Asn-382) and fifth (Asn-409) epidermal growth factor domains. The role of N-linked and simple O-linked carbohydrates in the functions of human thrombomodulin remain unclear. The present studies demonstrate, however, that the presence of chondroitin sulphate in human thrombomodulin has profound effects on all of the anticoagulant properties of this important anticoagulant thrombin receptor.

Antithrombotic agents from salivary glands of hematophagous animals.

The introduction of thrombolytic agents and adjunctive anticoagulant therapy in acute myocardial infarction and heparin-aspirin combinations in unstable angina have resulted in major gains in the acute management of these disorders; however, it is widely believed that available therapeutic agents, such as streptokinase and tissue plasminogen activator (t-PA), anticoagulants, such as heparin, and antiplatelet agents, such as aspirin, may not possess the optimal efficacy and safety profile. A major surge was initiated to identify alternative thrombolytic and antithrombotic drugs, and as a part of these efforts the isolation and characterization of protein salivary anticoagulants from hematophagous animal species has assumed importance. In the following, we briefly review these proteins and the development of peptides and peptidomimetic compounds based on their structures and discuss the potential utility of these compounds in cardiovascular disease therapy.

Relationship between post-translational glycosylation and anticoagulant function of secretable recombinant mutants of human thrombomodulin.

Two glycoforms of a soluble mutant of recombinant human thrombomodulin (rec.TM) were used to identify critical N- and O-linked glycans of the endothelial cell thrombin receptor. While N-linked glycans were not found to be involved in any function of rec.TM, an acidic chondroitin sulphate-like glycosaminoglycan (CSGAG) was found to be critical for all the direct anticoagulant functions of rec.TM, including inhibition of thrombin-mediated platelet aggregation. A glycoform of rec.TM lacking CSGAG had very poor anticoagulant activity. Furthermore, the glycoform of rec.TM possessing CSGAG showed strong inhibition by and had high affinity for poly-cationic basic proteins, whereas the CSGAG-deficient rec.TM did not. Monoclonal antibody binding as well as lectin mapping of rec.TM with agglutinins identified sialic acid containing O-linked glycans in both glycoforms additional to the CSGAG in high molecular weight rec.TM These findings define important molecular interactions modulating the anticoagulant function of TM, which appear to be critically regulated by CSGAG, and also showed that the overall post-translational glycosylation pattern of the two glycoforms was very similar except for the presence of CSGAG. The possibility exists that differently expressed glycoforms of TM may be crucial for the expression of endothelial cell-related anticoagulant potential in different vascular beds.

Different glycoforms of human thrombomodulin. Their glycosaminoglycan-dependent modulatory effects on thrombin inactivation by heparin cofactor II and antithrombin III.

The relationship between thrombomodulin-associated O-linked glycosammoglycans (GAGs) and the exogenous GAGs heparin or dermatan sulfate was studied in the inhibition of thrombin by antithrombin III (AT III) or heparin cofactor II (HC II). Both rabbit thrombomodulin (TM) and two glycoforms (a high-Mr form containing GAGs and a low-Mr form lacking the majority of O-linked GAGs) of a recombinant human TM deletion mutant (rec-TM) were used. The rapid inactivation of thrombin by HC II in the presence of dermatan sulfate was prevented by both the high-Mr rec-TM and the rabbit TM. In contrast, both rabbit TM treated with chondroitin ABC lyase to remove O-linked GAGs and the low-Mr form of rec-TM had only weak protecting effects. In the absence of exogeneous dermatan sulfate, thrombin inhibition by a high concentration of HC II was slightly accelerated by the high-Mr form of rec-TM but protected by rabbit TM. When thrombin inhibition by AT III in the presence of heparin was studied, both high-Mr rec-TM and rabbit TM again invoked a similar reduction of inactivation rates, whereas in the absence of exogenous heparin, both high-Mr forms accelerated thrombin inhibition by AT III. The diverse reactivities of various forms of TM towards HC II and AT III were also observed during protein C activation by the thrombin-TM complex. These results suggest that thrombin activity at the vessel wall or in fluid phase may undergo major kinetic modulations depending on the type of protease inhibitor, the presence or absence of exogenous GAGs and the glycosylation phenotype of TM. The dependence of TM anticoagulant function on the presence of an intrinsic GAG moiety suggests that variant glycoforms of this endothelial cell cofactor may be expressed differently in a species-, organ-, or tissue-specific manner as a means to regulate TM function in diverse vasculatures.

Disulfide pairing of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli.

The kringle-2 domain of tissue plasminogen activator, cloned and expressed in Escherichia coli (Wilhelm, O.G., Jaskunas, S.R., Vlahos, C.J., and Bang, N.U. (1990) J. Biol. Chem. 265, 14606-14611), was internally radiolabeled using [35S]methionine-cysteine. Following refolding and isolation, the labeled polypeptide was further purified by reverse-phase high performance liquid chromatography. The purified kringle-2 domain was digested with thermolysin, and the resulting peptides were purified by high performance liquid chromatography. Five major peptides containing 35S were obtained. Amino acid sequence analysis showed that these peptides represented various cleavage products containing one or more of the following disulfides: Cys180-Cys261, Cys201-Cys243, Cys232-Cys256 (sequence numbering based on Pennica et al. (Pennica, D., Holmes, W.E., Kohr, W.J., Hakins, R.N., Vehar, G. A., Ward, C.A., Bennett, W.F., Yelverton E., Seeburg, P.H., Heynecker, H.L., Goeddel, E.V., and Collen, D. (1983) Nature 301, 214-221)). These results confirm that the refolding methodology used produced kringle-2 with the predicted disulfide linkage and, thus, yielded material suitable for structural and functional studies.