The Amino Terminal Lectin-Like Domain of Thrombomodulin Is Required for Constitutive Endocytosis

Thrombomodulin (TM) is a multidomain protein that serves the endocytic process, deletant forms of TM lacking either the lectin-like region (residues 2-155) or the hydrophobic as a cofactor in a major natural anticoagulant system. To further characterize the structure-function of TM, we have region of the N-terminal domain (residues 161-202) were expressed in COS cells (COSdel.2-155 and COSdel.161-202, transfected COS cells with different truncated forms of TM. In the first form, COS cells expressing TM that lacks the respectively). Protein C cofactor activity was maintained in both cells. Although the COSdel.161-202 cells behaved simiputative signal peptide (17 residues); the lectin-like, hydrophobic N-terminal domain (226 residues); and 12 residues larly to wild-type TM-transfected cells, visual studies showed a lack of constitutive internalization of thrombinof the first epidermal growth factor (EGF)-like repeat (COSdel.238 cells) were found to function normally with reTM or anti-TM–TM complexes in the COSdel.2-155 cells. We conclude that the lectin-like domain of human TM serves to spect to TM transport to the cell surface and thrombin-dependent protein C activation. However, in contrast to wildregulate cell surface expression of TM via the endocytic route and therefore may also play a major physiologic role type TM, as visually studied by immunofluorescence and immunogold electron microscopy, the COSdel.238 cells did in controlling intracellular and extracellular accumulation of thrombin in a variety of biologic systems. not constitutively internalize anti-TM–TM or thrombin-TM complexes. To identify the region responsible for mediating q 1997 by The American Society of Hematology.

T site for thrombin and the last three of which are necessary for association and activation of protein C. The function of HROMBOMODULIN (TM) is a vascular endothelial cell receptor that is a critical cofactor in a major physiologically relevant natural anticoagulant system. 1,2 By en-the other EGF-like repeats is unknown. The third domain between the EGF-like repeats and the membrane-spanning hancing the activation of protein C by thrombin, TM leads to the cleavage and inactivation of factors V and VIII by region is rich in serines and threonines and contains four potential sites for O-linked glycosylation, to one of which activated protein C, thereby downregulating further production of thrombin. Functional and quantitative abnormalities is attached a chondroitin sulfate, which is important for full anticoagulant activity of TM. 2 Fourthly, there is a highly of protein C have commonly been reported to be associated with a hypercoagulable state 3,4 and recently there has been conserved transmembrane domain and, finally, a short cytoplasmic tail that contains potential sites of phosphorylation a family described with a mutation in the gene encoding TM that was associated with a thrombotic diathesis. 5 and a single cysteine that may mediate multimerization of the molecule. 16 Studies by several investigators have elucidated the putative structural organization of TM and the regions responsi-Considerable attention has been paid to identifying the structural determinants critical for normal anticoagulant ble for its anticoagulant function. [6][7][8][9][10][11] The mature single-chain glycoprotein in the human is 557 amino acids long and is function of TM. However, few studies have been directed towards elucidating the role of the other structural domains. structurally divided into five domains. The N-terminal region (residues 1-226) 10 has a module (residues 1-154) with homol- We have recently determined that TM undergoes constitutive endocytosis and that this process is not significantly affected ogy to the lectin domains of the hepatic asialoglycoprotein receptor 12 and IgE 13 as well as to members of the selectin by deletion of the cytoplasmic tail. 16, 17 The results suggested that alternative signals were necessary for the endocytic pro-family 14 and has two potential sites for N-linked glycosylation. From residues 155 to 226, there is a hydrophobic region cess to occur. In view of the fact that the N-terminal domain of TM has a hydrophobic region as well as a lectin-like that may be associated with the plasma membrane 15 and contains two potential sites for O-linked glycosylation. The module, we hypothesized that some or part of this region may interact with the plasma membrane and play a role in next domain is composed of six epidermal growth factor (EGF)-like repeats, the last two of which form the binding regulating intracellular trafficking of TM. To explore this possibility, we used COS cells to express human TM lacking the following: (1) the entire N-terminal domain of TM, (2) THROMBOMODULIN AND ITS LECTIN-LIKE DOMAIN 653 internalization and/or aggregation of TM. 17 CD71 is a murine mono-

RNA Isolation and Northern Analysis
clonal antihuman transferrin receptor antibody obtained from Sigma Total RNA was isolated from confluent cell monolayers by the (St Louis, MO). HL12-21 is a murine monoclonal antihuman factor method of Chomczynski and Sacchi. 20 Northern analyses and detec-IX antibody kindly provided by Dr E. Yeo (Toronto, Ontario, Cantions were performed as previously described. 21 The cDNA probe ada). Each of the antibodies was isotypically the same (IgG1k). used to detect TM mRNA was a Xho I-Xba I 3.1-kb fragment from Restriction enzymes were from Boehringer Mannheim Canada (Dorthe TM gene that spans the entire coding region for the protein. It val, Quebec, Canada), and radiolabeled products were from ICN was purified on a low melting point agarose gel followed by labeling Biomedicals, Mississauga, Ontario, Canada).
with a-32 P-dCTP using the random primer synthesis method. 22 The specific activity of the probe was 1 to 2 1 10 6 cpm/ng of DNA.

Vector Construction
Deletion of residues 017 to 238 from N-terminus of TM. Using Cell Culture the convenient restriction enzyme sites Xho I and Mlu I, a 950-bp COS-7 cells were grown in Dulbecco's modified Eagle's medium fragment of human TM cDNA that encodes the amino-terminal 256 (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 residues of nascent TM was removed from a 3,071-bp Xho I-BamHI mg/mL penicillin, and 100 mg/mL streptomycin. Transfected COS fragment of the TM DNA; the latter includes the entire coding region cells expressing the neomycin-resistant gene were selected and conand was previously subcloned into the plasmid Bluescript (pBS; tinually grown in the presence of 1 mg/mL Geneticin (GIBCO/ Stratagene, La Jolla, CA). 17 The Xho I and Mlu I sites of the re-BRL, Grand Island, NY). Cell cultures were incubated at 37ЊC in a maining linearized vector were filled in with dNTPs using Klenow humidified atmosphere of 5% CO 2 and 95% air, and media was and dephosphorylated with calf intestinal alkaline phosphatase changed 24 hours before all experiments. (Pharmacia, d'Urfe, Quebec, Canada). The Cla I linker [New England Biolabs, Beverly, MA; #1077 c(pCCCATCGATGGG)] containing an initiation codon was ligated to each end, resulting in Protein C Activation circularized DNA with the ATG codon in-frame for translation of The levels of functional cell surface TM were evaluated as prethe remainder of TM. After transformation of this recombinant plasviously reported 17 by incubating purified protein C and thrombin mid, the 2.1-kb Cla I-Xba I fragment was subcloned into Cla I-Xba I over the cells and quantitating the generation of activated protein C cut pBS and transformed into bacteria. Several colonies were picked, using the synthetic amidolytic substrate, S2238. Controls containing checked for the expected insert, and confirmed by DNA sequencing.
the same concentrations of thrombin and protein C in the absence An appropriate clone was selected and grown to large scale, and the of cells resulted in no activation of protein C. entire Xho I-Xba I insert coding for TM without the N-terminal 255 amino acids (5 of the initiation methionine) of the molecule (a Immunofluorescent Studies putative 17-residue signal peptide / 238 residues of mature TM) 10 Cells were grown on glass coverslip slides and washed three times was subsequently subcloned into the expression plasmid vector in phosphate-buffered saline (PBS). Cells were fixed with 3.0% pSR1neo 17 and introduced by electroporation into COS-7 cells to paraformaldehyde in 0.1 mol/L sodium phosphate pH 7.2, for 20 yield ''COSdel.238'' cells.
minutes and permeabilized with 0.2% Triton X-100 in PBS for 2 Deletion of lectin-like and hydrophobic regions of N-terminal minutes. After further washes in PBS, the cells were treated for 30 domain. The lectin-like module within the N-terminal domain of minutes with blocking solution (PBS with 1% bovine serum albu-TM was removed by recombination polymerase chain reaction min) and washed with PBS, and the first murine monoclonal antibody (PCR) in the following way. Oligonucleotide primers P1 (5CAAwas added at 10 mg/mL for 30 minutes at 37ЊC in blocking solution. CTCGAGCCCTGGCCGATCCGCAT) and P2(5ATTGCACGC-After further washes, detection was accomplished with rabbit-anti-GTGCTCGCAGC) that flank the region from nucleotide 1 to 959 19 mouse Fab bound to fluorescein isothiocyanate (FITC). Parallel and encoding the N-terminal domain of human TM were synthesized experiments using an irrelevant first antibody (HL12-21) were perfor the purposes of PCR amplification. Two primers (a 36-mer formed to exclude nonspecific immunofluorescence. [5GGCCTGGGGTTCCCCGCAACCTGCAGGCCACTGGCT] and its reverse complement) were constructed such that they overlapped and skipped the lectin-like module from residue 2 to 155, inclusive.

Conjugation of Proteins to Colloidal Gold
Each of these primers were matched with primers P2 and P1, respec-Colloidal gold with a mean diameter of 15 nm (SD Å 1.8), made tively, for PCR amplification, with the target DNA being pBS conusing the procedure of Frens, 23 was stably conjugated with thrombin taining the entire human TM cDNA (see above). The two PCR or murine monoclonal antibodies by methods previously reproducts of expected sizes 249 bp and 247 bp were purified, denaported. 16,17,24 The minimum amount of protein needed to ensure comtured, annealed, and amplified by PCR using P1 and P2 as the plete labeling of a given volume of colloidal gold solution was primers. The recombination product of 496 bp was digested with Xho established using the dilution-electrolyte flocculation test. 25 The gold I-Mlu I and subcloned to replace the wild-type N-terminal domain of conjugates were examined under electron microscopy (EM) before human TM in the expression vector pSR1neo. The hydrophobic use to ensure the absence of aggregates. module within the N-terminal domain of TM (residues 161-202, inclusive) was removed by similar techniques; however, the primer

Sample Preparation for Electron Microscopic Studies
(5CTTCCCAGCCACCTGCAGGCCCTTACAGCTAATGTGCAC) and its reverse complement were designed to delete the DNA encod-Before protein-gold labeling, cell layers were washed with ice cold serum-free media and incubated at 0ЊC for 40 minutes. Gold-ing the region from residues 161 to 202, inclusive. These oligonucleotide primers were paired, respectively, with primers P2 and P1 in labeled anti-TM antibody or thrombin was then added to a final concentration of 5 mg/mL and incubated for 60 minutes on ice. The PCR reactions to yield amplicons of 112 and 709 bp. The recombination product of 821 bp was then subcloned as described above into cell monolayers were washed with media and incubated for various times at 37ЊC, after which the cells were placed on ice, washed with Xho I-Mlu I-digested wild-type TM cDNA in pSR1neo. Both of the deletant constructs were confirmed by DNA sequencing, and the cold PBS, quickly scraped into a microfuge tube, and fixed with 2.5% glutaraldehyde in PBS. After postfixation in 1% osmium te-expression vectors were introduced into COS-7 cells by electroporation, resulting in COSdel.2-155 and COSdel.161-202 cells.
troxide and ethanol dehydration, the pellets were treated with propyl-For personal use only. on August 28, 2017. by guest www.bloodjournal.org From  transfected cell lines (Fig 2). or 3E2 specifically directed against human TM, followed by detection with FITC-secondarily labeled antibody (Figs 3

and 4). This confirmed that the deletant and wild-type forms Expression of Truncated TM by COS Cells
of TM could be synthesized, processed, and inserted into the plasma membrane and that it could associate with thrombin, COS cells have previously been stably transfected with protein C, and the specific monoclonal antibodies. No crosswild-type TM (COS.TM-CR cells) that express high cellreactivity with the irrelevant monoclonal antibody HL12-21 surface levels of a functional protein that is identical by (antihuman factor IX antibody) was detected. sodium dodecyl sulfate-polyacrylamide gel electrophoresis and endocytic routing to that from human umbilical vein endothelial cells (HUVEC). 17 For this report, we have trans-  tected in both cell lines (Figs 3A and 4A) by immunofluorescence and observed to be evenly distributed predominantly on the plasma membrane surface, with little evidence of intracellular localization. Cells were exposed to 37ЊC for 10 and 30 minutes to attempt to initiate the process of endocytosis. Under these conditions, internalization of the complexes was induced in the COS-TM.CR cells, as seen in Fig  3B and C. An intracellular punctate pattern was observed with a coincident decrease in intensity of the cell surface membrane fluorescence. The intracellular location was confirmed by parallel experiments in which permeabilization was not performed before fixation (Fig 3D). In this case, fluorescence was markedly diminished and restricted to the cell surface.
In contrast to the intracellular routing of TM in the COS-TM.CR cells, internalization of 24FM-TM complexes was not seen with the COSdel.238 cells (Fig 4). After exposure of the cells to 37ЊC for 10 and 30 minutes, the expression of TM was largely restricted to the cell surface and was  (Fig 4D).
Because of the possibility that either the signal peptide or the first EGF-like repeat of TM might be responsible for the observed effect on the endocytosis of the molecule, we repeated the above studies with COS cells expressing deletant forms of TM that either lacked only the lectin-like module (residues 2-155; COSdel.2-155 cells) or the hydrophobic region from residues 161 to 202 (COSdel.161-202 cells; Fig  1). Transcription of the expected size mRNAs in each cell line was confirmed by Northern blot analyses, and expression of functional cell-surface TM was verified by determination of thrombin-dependent protein C activity (Table 1).
After preincubation of the COSdel.2-155 cells on ice with 24FM antibodies, we examined the endocytic role of the lectin-like module of TM by increasing the temperature to 37ЊC for varying periods of time. As with the COSdel.238 cells, there was almost no evidence of internalization of the antibody-TM complexes over a 30-minute period (Fig 5). Furthermore, the intensity of surface labeling on the cells between residues 161 and 202 was similarly examined by immunofluorescence studies using the COSdel.161-202 cells. In contrast to the effect of deleting residues 2 to 155, there was no apparent effect on internalization of TM when residues 161-202 were deleted. Using the 24FM antibodies, exposure of the COSdel.161-202 cells to 37ЊC for 10 and 30 minutes resulted in rapid internalization of the antibody-TM complexes in a pattern apparently unchanged from that seen with the wild-type COS.TM-CR cells (data not shown).

Electron Microscopy Studies
When the COS-TM.CR cells were incubated at 4ЊC with the 24FM-gold complexes, gold particles became randomly distributed on the cell membrane surface as individual particles or in clusters of 2 or 3 ( Fig 6A). Controls using 24FMgold complexes incubated with either COS cells or pSR1neotransfected cells or an irrelevant gold-complexed antibody (HL12-21) incubated with the COS.TM-CR or COSdel.238 cells resulted in an absence of binding to the cell surface. As shown previously, 17 internalization of specific anti-TM gold particles in the COS-TM.CR cells was evident as the cells were exposed to 37ЊC for varying periods of time ( Fig  6B through D). This was characterized by redistribution of many of the particles into clusters and progressive accumulation into mostly noncoated plasma membrane invaginations, pits, and vesicles. By 10 to 20 minutes, gold particles were  .238 cells (Fig 4), there was no evidence of internalization or shedding of the complex. reported 17 using the same immunogold complexes on non-For personal use only. on August 28, 2017. by guest www.bloodjournal.org From complexed monoclonal anti-TM antibody (3E2) known to identify a different epitope than 24FM, the above experiments were repeated with identical results, in that constitutive endocytosis was not seen using the COSdel.2-155 cells, whereas it was readily apparent in the COS.TM-CR cells. There was no discernible difference in the endocytic routing of gold-complexes of 24FM or thrombin in COSdel.161-202 and COS.TM-CR cells, further illustrating the specificity of the region (residues 2-155) that is critical for internalization of TM.

DISCUSSION
In ongoing studies to characterize the structure-function correlations of thrombomodulin, we have recently shown that the cytoplasmic tail of the molecule is not required for efficient constitutive endocytosis via non-clathrin-coated pits, yet it may be important both for clathrin-coated pitmediated endocytosis and for multimerization of TM. 16,17 The mechanism by which TM might be internalized without a cytoplasmic tail remained a mystery and prompted us to investigate other regions of TM. Previous studies have suggested that the nonpolar amino terminus end of TM that has lectin-like homology and a hydrophobic region may be

Fig 7. EM localization of gold-conjugated thrombin in COS.TM-CR cells. After preincubation of cells with gold-complexed thrombin,
associated with the plasma membrane surface. 15 We hypoththe cells were exposed to 37ЊC for 0, 10 copy, was strong evidence that the lectin-like module of TM, Routing of gold-labeled thrombin was also re-examined which contains two potential sites for N-linked glycosylain the COS-TM.CR and the results, depicted in Fig 7, are tion, was critical in mediating its constitutive endocytic routconsistent with those previously reported by us. 16,17 Without ing from the cell surface membrane. Future similar studies induction of internalization, the thrombin-gold complexes with more subtle mutations will determine whether the obwere seen at the cell surface singly and in clusters of 1 or served findings are dependent on the lectin-like properties 2 ( Fig 7A). As endocytosis was induced for 10 to 45 minutes, of this domain, its glycosylation, or, alternatively, due to internalization of the gold was evident predominantly via tertiary structural changes in other regions of the molecule noncoated pits and vesicles, with ultimate localization in that may have been induced by the deletion of this module. multivesicular bodies and lysosomes (Fig 7B and C).
The deletant form of TM expressed by the COSdel.238 The effects of exposing either the COSdel.238 cells or the cells was initially constructed using convenient restriction COSdel.2-155 cells to 37ЊC on the routing of 24FM-gold enzyme sites. Not only was the entire N-terminal domain and thrombin-gold complexes were similarly studied (Figs removed, but the recombinant deletant TM was also lacking 8 through 11). Before induction of internalization, the 24FMthe putative signal peptide and 12 residues of the first EGFgold (Figs 8A and 10A) and thrombin-gold (Figs 9A and like domain. It was interesting to note that, even without a 11A) complexes distributed singly or in small clusters exclusignal peptide, TM could be transported intact to the cell sively on the cell surface in a pattern similar to that seen surface. Because N-terminal amino acid sequencing of nawith the COS-TM.CR cells. However, in marked contrast to scent TM has not been reported, it is possible that there is the intracellular pathway characterized by the COS.TM-CR no functional or cleavable signal peptide. Indeed, it has been cells, internalization of the gold complexes was notably abnoted that the putative signal peptide lacks those basic amino sent in both the COSdel.238 (Figs 8 and 9) and COSdel.2acid residues often marking each end of such sequences. 6 155 cells (Figs 10 and 11). With incubation of these cells at Transmembrane proteins that lack typical N-terminal signal 37ЊC for 10 to 45 minutes, complexes of 24FM-gold (Figs peptides are not uncommon and include, for example, the 8B through D and 10B through D) and thrombin-gold (Figs asialoglycoprotein receptor, 27 the transferrin receptor, 28 hep-9B and C and 11B and C) were almost exclusively seen on sin, 29 and the effector protease receptor-1, 30 all of which are the cell surface, whereas intracellular particles represented able to insert correctly across the membrane, presumably less than 2% of all gold particles. Empty clathrin-coated and noncoated vesicles were easily identified. Using a gold-due to their possessing a hydrophobic domain. Thus, it is not For personal use only. on August 28, 2017. by guest www.bloodjournal.org From  Fig 5 and exposed to  37ЊC for 0, 10, 30, and 45 minutes (A, B, C, and D,  respectively). In contrast to what is seen in Fig 5, gold particles remained on the cell surface (arrowheads), although clusters formed within 10 minutes of exposure to 37ЊC. Rare intracellular particles are seen (arrow).
surprising that recombinant TM synthesized by COSdel.238 plexes and complement the heat shock-induced transcriptional upregulatory effects on TM hypothesized to occur in cells was transported to the cell surface apparently normally.
The in vitro expression of TM is regulated by both tran-vivo during inflammation and infection, 38 possibly as a vasoprotective measure. Finally, cell/tissue-specific expression of scriptional and posttranscriptional mechanisms (for review, see Hirokawa and Aoki 31 ), with considerable variability carbohydrates or lectins that bind to TM's lectin module or glycosaminoglycans within that module, respectively, may noted between cell and tissue types. For example, there is evidence that internalization of thrombin-TM does not occur result in differential properties of TM with respect to its ability to undergo constitutive endocytosis. This may be im-in human saphenous vein endothelial cells, [31][32][33] whereas in HUVEC, bovine aortic endothelial cells, A549 cells, and portant in the low-flow circulation of saphenous veins in which continuous expression of cell-surface TM is im-wild-type TM-transfected COS cells, endocytosis of thrombin-TM is well documented. 17,[31][32][33][34][35][36] There are several mecha-portant; whereas, in high-flow vascular beds, the response to injury may be critical, and suppression of cell-surface TM nisms by which the lectin-like module of TM may contribute to the variability in its expression between cells and/or tis-may be important to allow appropriate local hemostasis and eventual healing. sues. For example, changes in N-linked glycosylation within the lectin-like region may be cell/tissue specific and/or may In addition to that of TM, the lectin domains of receptors on, eg, activated macrophages, hepatocytes, and fibroblasts have be altered during in vivo stresses, preventing the molecule from being internalized, and thereby serving to maintain also been implicated in regulating receptor-mediated endocytosis of glycoconjugates. [39][40][41] The mechanism(s) of initiation of thromboresistance. Alternatively, the release of soluble forms of TM (containing all of the extracellular domains) these processes is also largely speculative and the focus of considerable investigation. Although our studies support a hy-that occurs during inflammation and a variety of vascular disorders 37 may accumulate in regions of the vasculature and pothesis that the lectin-like properties of TM may mediate its internalization, it is equally possible that signaling via this region saturate the binding sites of an as yet unidentified carbohydrate ligand that may otherwise interact with the intact cell in response to a stimulus (such as following the association of TM with thrombin and/or protein C) and unrelated to its lectin-surface TM lectin module and induce internalization of the molecule. This competitive effect would then result in the homology leads to transmission of information required for initiation of the steps necessary for endocytosis to occur. prevention of constitutive endocytosis of thrombin-TM com- For personal use only. on August 28, 2017. by guest www.bloodjournal.org From and recycled or degraded. The fate of thrombin is unknown, but it appears that each thrombin molecule is restricted to activating a limited number of thrombin receptors. This led to the conclusion that, for thrombin to become inaccessible to other thrombin receptors, it must (1) be similarly internalized via the activated thrombin receptor, (2) become neutralized by thrombin inhibitors, or (3) associate with other thrombin receptors, such as TM. 54 The recent in vivo thrombin receptor gene-inactivation study 55 supports the existence of at least one other thrombin receptor. There is currently no evidence to indicate that thrombin is internalized via the thrombin receptor and, although serine protease inhibitors such as anti-thrombin III and protease nexin I may neutralize thrombin, it is likely that TM plays a critical role in mopping up the excess thrombin, rendering it inaccessible to further activation of the thrombin receptor.
The extent of the potential importance of TM in regulating thrombin's diverse actions is illustrated by the fact that the expression of TM is not restricted to the vascular endothelium. Rather, it is also synthesized by vascular smooth muscle cells, synovial lining cells, osteoblasts, syncytiotrophoblasts, keratinocytes, cells of the central nervous system, and several hematopoietic cells. 21,56-59 The developmental expression of TM in both vascular tissues and at sites of cell- main of TM is critical for directing the molecule intracellularly from the membrane surface. Although the in vivo significance of this process is currently unknown, it is worth Beyond thrombin's action to cleave fibrinogen to generate noting that lectins and their corresponding carbohydrate lia fibrin clot and to activate protein C when associated with gands may contribute to a variety of physiologically imone of its major receptors, the serine protease has several portant processes including tumor cell recognition, cell adheprofound biologic effects on a variety of cells and organ sion, cell activation, signal transduction, differentiation, and systems. Thrombin activates platelets, inducing phosphoinoapoptosis. 63 In vivo studies using targeted deletion strategies sitide hydrolysis, eicosanoid formation, and protein phosphorylation, leading to adhesion, aggregation, and secretion. Vascular endothelial cell expression and/or release of nitric oxide, 42 platelet-activating factor, 43 tissue-type plasminogen activator, plasminogen activator inhibitor-1, and urokinasetype plasminogen activator have been reported to be augmented by thrombin, 44 whereas urokinase receptor (u-PAR) may be decreased in endothelial cells 45 or increased in smooth muscle cells. 46 Furthermore, adhesion molecules may be upregulated by thrombin, promoting neutrophil adhesion, and thus contribute to a proinflammatory response. Thrombin also has mitogenic activity on a variety of cells, including lymphocytes, fibroblasts, macrophages, mesangial cells, neuroblastoma cells, and osteoblasts. 44,[47][48][49][50] Finally, in the inflamed joint, thrombin has recently been implicated in the pathology of arthritis by inducing proteoglycan release in the degradation of cartilage. 51 Many of the above-mentioned biologic actions of throm- gests that thrombin binds to and cleaves the N-terminus of respectively). Gold particles remained distributed on the cell surface the thrombin receptor, exposing a so-called tethered ligand during this period (arrowheads), with only a rare intracellular particle found.
that activates the receptor, after which it may be internalized For personal use only. on August 28, 2017. by guest www.bloodjournal.org From occurs via clathrin-coated and non-coated pits. J Cell Physiol in mice will eventually provide us with further insights into 151:604, 1992 the structure-function correlates of TM not only in adult 18. Conway EM, Lau HK, Bauer KA, Rosenberg RD: Developpathophysiology, but also in normal development. ment of a radioimmunoassay for quantitating prethrombin 2 in human plasma. J Lab Clin Med 110:567, 1987