Title: Role of Thrombospondin-1 in Control of von Willebrand Factor Multimer Size in Mice
Abstract: Plasma von Willebrand factor (VWF) is a multimeric glycoprotein from endothelial cells and platelets that mediates adhesion of platelets to sites of vascular injury. In the shear force of flowing blood, however, only the very large VWF multimers are effective in capturing platelets. The multimeric size of VWF can be controlled by proteolysis at the Tyr842-Met843 peptide bond by ADAMTS13 or cleavage of the disulfide bonds that hold VWF multimers together by thrombospondin-1 (TSP-1). The average multimer size of plasma VWF in TSP-1 null mice was significantly smaller than in wild type mice. In addition, the multimer size of VWF released from endothelium in vivo was reduced more rapidly in TSP-1 null mice than in wild type mice. TSP-1, like ADAMTS13, bound to the VWF A3 domain. TSP-1 in the wild type mice, therefore, may compete with ADAMTS13 for interaction with the A3 domain and slow the rate of VWF proteolysis. TSP-1 is stored in platelet α-granules and is released upon platelet activation. Significantly, platelet VWF multimer size was reduced upon lysis or activation of wild type murine platelets but not TSP-1 null platelets. This difference had functional consequences in that there was an increase in collagen- and VWF-mediated aggregation of the TSP-1 null platelets under both static and shear conditions. These findings indicate that TSP-1 influences plasma and platelet VWF multimeric size differently and may be more relevant for control of the VWF released from platelets. Plasma von Willebrand factor (VWF) is a multimeric glycoprotein from endothelial cells and platelets that mediates adhesion of platelets to sites of vascular injury. In the shear force of flowing blood, however, only the very large VWF multimers are effective in capturing platelets. The multimeric size of VWF can be controlled by proteolysis at the Tyr842-Met843 peptide bond by ADAMTS13 or cleavage of the disulfide bonds that hold VWF multimers together by thrombospondin-1 (TSP-1). The average multimer size of plasma VWF in TSP-1 null mice was significantly smaller than in wild type mice. In addition, the multimer size of VWF released from endothelium in vivo was reduced more rapidly in TSP-1 null mice than in wild type mice. TSP-1, like ADAMTS13, bound to the VWF A3 domain. TSP-1 in the wild type mice, therefore, may compete with ADAMTS13 for interaction with the A3 domain and slow the rate of VWF proteolysis. TSP-1 is stored in platelet α-granules and is released upon platelet activation. Significantly, platelet VWF multimer size was reduced upon lysis or activation of wild type murine platelets but not TSP-1 null platelets. This difference had functional consequences in that there was an increase in collagen- and VWF-mediated aggregation of the TSP-1 null platelets under both static and shear conditions. These findings indicate that TSP-1 influences plasma and platelet VWF multimeric size differently and may be more relevant for control of the VWF released from platelets. von Willebrand factor (VWF) 1The abbreviations used are: VWF, von Willebrand factor; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; BSA, bovine serum albumin; CBA, collagen binding affinity; ELISA, enzyme-linked immunosorbant assay; NEM, N-ethylmaleimide; TSP-1, thrombospondin-1; mAb, monoclonal antibody; Pipes, 1,4-piperazinediethanesulfonic acid. serves a critical role in hemostasis by facilitating the initial tethering of platelets to the subendothelium at high shear (reviewed in Ref. 1Sadler J.E. Annu. Rev. Biochem. 1998; 67: 395-424Crossref PubMed Scopus (1138) Google Scholar). VWF is synthesized by megakaryocytes and endothelial cells and circulates in blood as a series of multimers made up of a variable number of disulfide-linked 500-kDa homodimers (2Counts R.B. Paskell S.L. Elgee S.K. J. Clin. Invest. 1978; 62: 702-709Crossref PubMed Scopus (88) Google Scholar). Only the larger multimers of VWF are effective in hemostasis, since a selective deficiency of these forms is associated with a bleeding diathesis, type IIA von Willebrand disease (3Furlan M. Ann. Hematol. 1996; 72: 341-348Crossref PubMed Scopus (190) Google Scholar). The intracellular assembly of VWF multimers is a stepwise process. First, individual pro-VWF subunits are linked in a tail-to-tail orientation by C-terminal disulfide bonds to form pro-VWF dimers, which are then linked in a head-to-head orientation by N-terminal disulfide bonds to form VWF multimers. The largest VWF multimers have a molecular mass in excess of 20,000 kDa (reviewed in Ref. 4Wagner D.D. Annu. Rev. Cell Biol. 1990; 6: 217-246Crossref PubMed Google Scholar). The VWF secreted constitutively from endothelial cells is composed of dimers and small multimers in contrast to the ultralarge multimers that are released from the storage compartment (5Sporn L.A. Marder V.J. Wagner D.D. Cell. 1986; 46: 185-190Abstract Full Text PDF PubMed Scopus (343) Google Scholar). The Weibel-Palade bodies in endothelial cells and the α-granules of platelets release ultralarge VWF in response to vascular injury and platelet activation. The largest VWF multimers in plasma are smaller than those stored within endothelial cells and platelets (6Moake J.L. Rudy C.K. Troll J.H. Weinstein M.J. Colannino N.M. Azocar J. Seder R.H. Hong S.L. Deykin D. N. Engl. J. Med. 1982; 307: 1432-1435Crossref PubMed Scopus (923) Google Scholar). Two mechanisms operate in regulating VWF multimer size: shear-dependent hydrolysis of VWF multimers by the VWF-cleaving metalloproteinase, ADAMTS13 (7Furlan M. Robles R. Lamie B. Blood. 1996; 87: 4223-4234Crossref PubMed Google Scholar, 8Tsai H.M. Blood. 1996; 87: 4235-4244Crossref PubMed Google Scholar, 9Fujikawa K. Suzuki H. McMullen B. Chung D. Blood. 2001; 98: 1662-1666Crossref PubMed Scopus (516) Google Scholar) (a disintegrin-like and metalloprotease with thrombospondin type 1 motif) and cleavage of the linking disulfides by the plasma and platelet glycoprotein, thrombospondin-1 (10Xie L. Chesterman C.N. Hogg P.J. J. Exp. Med. 2001; 193: 1341-1349Crossref PubMed Scopus (112) Google Scholar). A severe deficiency of ADAMTS13 is associated with congenital and acquired forms of thrombotic thrombocytopenic purpura (11Levy G.G. Nichols W.C. Lian E.C. Foroud T. McClintick J.N. McGee B.M. Yang A.Y. Siemieniak D.R. Stark K.R. Gruppo R. Sarode R. Shurin S.B. Chandrasekaran V. Stabler S.P. Sabio H. Bouhassira E.E. Upshaw Jr., J.D. Ginsburg D. Tsai H.M. Nature. 2001; 413: 488-494Crossref PubMed Scopus (1451) Google Scholar, 12Furlan M. Robles R. Galbusera M. Remuzzi G. Kyrle P.A. Brenner B. Krause M. Scharrer I. Aumann V. Mittler U. Solenthaler M. Lammle B. N. Engl. J. Med. 1998; 339: 1578-1584Crossref PubMed Scopus (1486) Google Scholar, 13Tsai H.M. Lian E.C. N. Engl. J. Med. 1998; 339: 1585-1594Crossref PubMed Scopus (1493) Google Scholar), a disorder characterized by a schistocytic hemolytic anemia, a consumptive thrombocytopenia, and variable degrees of renal and neurological impairment. The persistence of unprocessed ultralarge VWF multimers in the circulation is thought to precipitate platelet clumping in arterioles and capillaries, resulting in tissue ischemia (6Moake J.L. Rudy C.K. Troll J.H. Weinstein M.J. Colannino N.M. Azocar J. Seder R.H. Hong S.L. Deykin D. N. Engl. J. Med. 1982; 307: 1432-1435Crossref PubMed Scopus (923) Google Scholar). The full-length ADAMTS13 transcript is expressed predominantly in the liver (11Levy G.G. Nichols W.C. Lian E.C. Foroud T. McClintick J.N. McGee B.M. Yang A.Y. Siemieniak D.R. Stark K.R. Gruppo R. Sarode R. Shurin S.B. Chandrasekaran V. Stabler S.P. Sabio H. Bouhassira E.E. Upshaw Jr., J.D. Ginsburg D. Tsai H.M. Nature. 2001; 413: 488-494Crossref PubMed Scopus (1451) Google Scholar, 14Zheng X. Chung D. Takayama T.K. Majerus E.M. Sadler J.E. Fujikawa K. J. Biol. Chem. 2001; 276: 41059-41063Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar) and appears to cleave ultralarge VWF multimers as they are secreted from endothelial cells and undergo conformational change when exposed to high shear (15Dong J.F. Moake J.L. Nolasco L. Bernardo A. Arceneaux W. Shrimpton C.N. Schade A.J. McIntire L.V. Fujikawa K. Lopez J.A. Blood. 2002; 100: 4033-4039Crossref PubMed Scopus (629) Google Scholar). The thrombospondins are a family of extracellular glycoproteins that function in cell-cell and cell-matrix communication and modulate cellular phenotype (16Lawler J. Curr. Opin. Cell Biol. 2000; 12: 634-640Crossref PubMed Scopus (377) Google Scholar). We reported that the supernatant of cultured endothelial cells possessed VWF reductase activity and identified TSP-1 as a VWF reductase (10Xie L. Chesterman C.N. Hogg P.J. J. Exp. Med. 2001; 193: 1341-1349Crossref PubMed Scopus (112) Google Scholar, 17Xie L. Chesterman C.N. Hogg P.J. Thromb. Haemostasis. 2000; 84: 506-513Crossref PubMed Scopus (40) Google Scholar). More recently, we showed that the VWF-reducing activity of TSP-1 centers on a free thiol at Cys974 in the Ca2+-binding C-terminal sequence of the protein (18Pimanda J.E. Annis D.S. Raftery M. Mosher D.F. Chesterman C.N. Hogg P.J. Blood. 2002; 100: 2832-2838Crossref PubMed Scopus (31) Google Scholar). The role of TSP-1 in regulating VWF multimer size in vivo and its relationship to ADAMTS13 activity was unknown. To clarify these issues, we have characterized the plasma and platelet VWF multimer pattern in TSP-1-/- and TSP-1+/+ C57BL/6 mice. Incubation of VWF with TSP-1 in vitro results in smaller VWF multimers. Surprisingly, TSP-1 contributes to the persistence of larger VWF multimers in the circulation possibly by negatively regulating ADAMTS13 activity. On the other hand, platelet VWF multimer size was reduced upon lysis or activation of wild type platelets but not TSP-1 null platelets. This difference has functional relevance as the TSP-1 null platelets exhibited an increase in collagen- and VWF-mediated aggregation under static and shear conditions. We discuss the implications of these findings with regard to the initiation and development of an arterial thrombus. Proteins and Reagents—Leupeptin, d-Phe-Pro-Arg-chloromethyl ketone, and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) were from Calbiochem, and bovine thrombin, N-ethylmaleimide (NEM), EDTA, 1,10-phenanthroline, phenylmethanesulfonyl fluoride, Triton X-100, and Tween 20 were from Sigma. Aprotinin was from Bayer AG (Leverkusen, Germany), and 1-desamino-8-d-arginine vasopressin (desmopressin) was from Ferring AB (Limhamn, Sweden). TSP-1 was purified from human platelet concentrates (19Murphy-Ullrich J.E. Mosher D.F. Blood. 1985; 66: 1098-1104Crossref PubMed Google Scholar); the recombinant TSP-1 fragments, CP123-1 (residues 294-529) and E3CaG-1 (residues 630-1152), were expressed in insect cells and were a gift from Dr. Deane Mosher (20Mosher D.F. Huwiler K.G. Misenheimer T.M. Annis D.S. Methods Cell Biol. 2002; 69: 69-81Crossref PubMed Scopus (40) Google Scholar). The residue numbers are for the mature protein. The recombinant VWF A3 domain was expressed in E. coli and purified from bacterial inclusion bodies and was a gift from Dr. Miguel Cruz (21Cruz M.A. Yuan H. Lee J.R. Wise R.J. Handin R.I. J. Biol. Chem. 1995; 270: 19668Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The anti-TSP-1 monoclonal antibody, mAb 133, was a gift from Dr. Joanne Murphy-Ullrich, and HB8432 was produced from a murine hybridoma cell line obtained from American Type Culture Collection (Manassas, VA). All other reagents were of analytical grade. Animals—Wild type (TSP-1+/+) and TSP-1 null (TSP-1-/-) C57BL/6 mice were used in this study. TSP-1-/- mice (22Lawler J. Sunday M. Thibert V. Duquette M. George E.L. Rayburn H. Hynes R.O. J. Clin. Invest. 1998; 101: 982-992Crossref PubMed Scopus (387) Google Scholar) were generated in Dr. Jack Lawler's laboratory (Boston, MA) by homologous recombination in 129/Sv-derived ES cells implanted in C57BL/6 blastocysts. Chimeras were bred to C57BL/6 mice, the offspring were genotyped, and heterozygotes were back-crossed (N9) to a C57Bl/6 genetic background (99.6%). A breeding program was established at the biological resource center at the University of New South Wales (Sydney, Australia), and animals aged between 6 weeks and 6 months and of both sexes were used. The experimental procedures were approved by the Animal Care and Ethics Committee of the University of New South Wales. Preparation of Plasma—The mice were anesthetized with isoflurane (Abbott), and blood was collected by cardiac puncture using a 1-ml syringe and a 26-gauge needle and mixed with 3.8% trisodium citrate anticoagulant in a 6:1 (v/v) ratio. For the desmopressin time course study, free flowing blood from the saphenous vein was collected via heparin-coated capillary tubes and mixed with citrate anticoagulant as above. To evaluate human plasma and platelet VWF, blood was collected by venepuncture using an 18-gauge needle and a 20-ml syringe and mixed with citrate anticoagulant. Platelet-rich plasma was obtained by centrifuging blood at 150 × g for 20 min. The platelet-rich plasma was then spun at 500 × g for a further 20 min to obtain a platelet pellet. The supernatant was recentrifuged at 2000 × g for a further 15 min to prepare platelet poor plasma. A platelet count was performed to confirm the absence of platelets, and aliquots of platelet-poor plasma were frozen at -80 °C till further use. For the experiments using pooled mouse plasma, equal volumes of blood were pooled prior to centrifugation. Preparation of Washed Platelets—The platelets obtained from platelet rich plasma were washed three times with 5 mm Pipes, pH 6.8, buffer containing 0.145 m NaCl, 4 mm KCl, 0.5 mm Na2HPO4, 1 mm MgCl2, and 5 mm dextrose. The platelet pellet after the final wash was resuspended in 20 mm Hepes, pH 7.4, buffer containing 0.137 m NaCl, 4mm KCl, 0.5 mm Na2HPO4, 0.1 mm CaCl2,and 5 mm dextrose (platelet Hepes), and the platelet count was adjusted to 2 × 109/ml. Preparation of a Platelet Lysate—Suspensions of washed platelets were centrifuged at 7000 × g for 2 min, and the platelet pellet was resuspended in 20 mm Hepes, pH 7.4, buffer containing 0.14 m NaCl, 1% Triton X-100, 0.05% Tween 20, 10 μm leupeptin, 2 mm 1,10-phenanthroline, phenylmethanesulfonyl fluoride, 1 mm AEBSF, 1 mm phenanthroline, and 10 μm aprotinin (lysis buffer) and incubated at 37 °C for 30 min. 5 mm EDTA and 10 mm NEM were added to the lysis buffer either before or 5 min into the incubation period. The suspensions of lysed platelets were then centrifuged at 7000 × g for 2 min, and aliquots of the supernatant were stored at -80 °C until further use. Thrombin Activation of Platelets and Preparation of Platelet Releasate—A suspension of washed platelets was preincubated at 37 °C for 30 min. Aggregation was performed at 37 °C by adding 1 unit·ml-1 thrombin to a continuously stirred suspension of platelets. The platelets were stirred for a total of 10 min with the addition of 5 μm d-Phe-Pro-Argchloromethyl ketone, 10 μm leupeptin, 1 mm AEBSF, 1 mm phenanthroline, 2 mm 1,10-phenanthroline, phenylmethanesulfonyl fluoride, and 10 μm aprotinin at 2 min and 5 mm EDTA and 10 mm NEM at 5 min. The suspension was centrifuged at 7000 × g for 2 min, and aliquots of the supernatant were stored at -80 °C until further use. The aggregated platelet pellet was then lysed with lysis buffer containing 5 mm EDTA and 10 mm NEM and incubated at 37 °C for 30 min. The lysed platelets were then centrifuged at 7000 × g for 2 min, and aliquots of the supernatant were stored at -80 °C. The Measurement of VWF Multimer Size—Two measurements of multimer size were performed: agarose gel electrophoresis and collagen binding. The samples were assayed using an in-house method at the Royal Prince Alfred Hospital (Sydney, Australia) modified from Ruggeri et al. (23Ruggeri Z.M. Zimmerman T.S. J. Clin. Invest. 1980; 65: 1318-1325Crossref PubMed Scopus (358) Google Scholar). Briefly, aliquots of murine and human plasma, platelet lysate, and releasate were diluted 10-fold in a 200 mm Tris, pH 6.8, buffer containing 10% glycerol, 1% SDS, 2 mm EDTA, and 0.01% bromphenol blue and warmed to 56 °C for 20 min. 20 μl of the mix was loaded onto a 1% agarose gel (Seakem™ HGT(P)-agarose; Cambrex, Santa Rosa, CA) in Tris-glycine-SDS electrophoresis buffer, pH 8.3, mounted on a Protean™ II xi cell system (Bio-Rad), and electrophoresed at 4 °C at 125 V for 5 h. The gels were fixed, and the nonspecific binding sites were blocked with skim milk, followed by an overnight incubation with 125I-labeled anti-human VWF polyclonal antibodies (Dako, Carpinteria, CA). The gels were then washed in distilled water, dried on GelBond film (BMA, Rockland, ME), and exposed to Eastman Kodak Co. BIOMAX MS film. The autoradiographs were developed at 48 h. Densitometry was performed using a Fluor-S MultiImager and Quantity One software from Bio-Rad. Aliquots of murine plasma, platelet lysate, and platelet releasate were diluted 10-fold in 20 mm imidazole, pH 7.3, buffer containing 0.12 m NaCl, 5 mm citric acid (ELISA buffer) and 5% bovine serum albumin (BSA), and the VWF collagen binding affinity and VWF antigen level were each assayed in triplicate as described by Favaloro et al. (24Favaloro E.J. Grispo L. Exner T. Koutts J. Blood Coagul. Fibrinolysis. 1991; 2: 285-291Crossref PubMed Scopus (106) Google Scholar). Murine VWF was detected using a 1000-fold dilution of peroxidase-conjugated anti-human VWF polyclonal antibodies (Dako, Carpinteria, CA) in ELISA buffer containing 0.1% BSA. The human samples were diluted 100-fold in ELISA buffer containing 5% BSA. The OD values of the ELISA plate were monitored in the plate reader, and the reactions were stopped at 0.2-0.3 OD rather than at a fixed time point for improved accuracy of the individual measurements. Although this results in a variation of the collagen binding affinity (CBA)/VWFAg ratio of plasma and platelet VWF between experiments, this was considered less important than the accuracy of each measurement for a given experiment. Binding of TSP-1 to VWF A3 Domain—VWF A3 domain (1 μm solution in 0.1 m NaHCO3, pH 7.3) was adsorbed to Nunc PolySorp 96-well plates overnight at 4 °C in a humid environment. Wells were washed three times with ELISA buffer plus 0.1% BSA, and nonspecific binding sites were blocked by incubating wells with 200 μl of ELISA buffer plus 5% BSA for 90 min at 37 °C and then washed twice with ELISA buffer plus 0.1% BSA. Dilutions of TSP-1 or the E3CaG-1 fragment were prepared in 50 mm Hepes buffer, pH 7.4, containing 0.14 m NaCl, 1 mm CaCl2,1 mg·ml-1 polyethylene glycol 6000 and 1% BSA. Reactions were in 100 μl for 60 min at room temperature with orbital shaking. Wells were washed four times with ELISA buffer plus 0.1% BSA and incubated with 100 μl of 5 μg·ml-1 mAb 133 for 60 min at room temperature with orbital shaking. Wells were washed three times with ELISA buffer plus 0.1% BSA and incubated with 100 μl of a 1:1000 dilution of rabbit anti-mouse peroxidase-conjugated antibodies for 30 min at room temperature with orbital shaking. Wells were washed three times with ELISA buffer plus 0.1% BSA, and the peroxidase was detected as previously described (24Favaloro E.J. Grispo L. Exner T. Koutts J. Blood Coagul. Fibrinolysis. 1991; 2: 285-291Crossref PubMed Scopus (106) Google Scholar). mAb 133 recognizes an epitope within E3CaG-1. To detect binding of TSP-1 to VWF A3 in the presence of CP123-1 or E3CaG-1, HB8432 was substituted for mAb 133. HB8432 recognizes an epitope within the epidermal growth factor-like domains of TSP-1 (25Prater C.A. Plotkin J. Jaye D. Frazier W.A. J. Cell Biol. 1991; 112: 1031-1040Crossref PubMed Scopus (188) Google Scholar) and does not react with either CP123-1 or E3CaG-1 (data not shown). Static Platelet Aggregometry—Pooled platelet-rich plasma was prepared as described, and the platelet count was adjusted to 300 × 109/liter using platelet-poor plasma. TSP-1+/+ and TSP-1-/- mouse platelet-rich plasmas were incubated at 37 °C for 10 min, and the platelet aggregation response to SKF Horm™ collagen (Nycomed, Ismaning, Munchen) and ADP (Chrono-Log Corp., Havertown, PA) was measured over 10 min in a platelet aggregometer (Chrono-Log Corp.). The role of VWF in the formation of a platelet aggregate was investigated by preincubating TSP-1-/- mouse platelet-rich plasma with rabbit anti-human VWF polyclonal antibodies (DAKO, Carpinteria, CA) or control normal rabbit immunoglobulin at 37 °C for 30 min prior to the addition of collagen. Shear-induced Platelet Aggregometry—1 ml of whole blood was collected by cardiac puncture from six 12-week-old female TSP-1+/+ and TSP-1-/- mice and added to 140 μl of citrate-phosphate-dextrose anticoagulant. Shear-induced platelet aggregation was performed using a PFA-100™ test system (26Kundu S.K. Heilmann E.J. Sio R. Garcia C. Davidson R.M. Ostgaard R.A. Semin. Thromb. Hemost. 1995; 21: 106-112PubMed Google Scholar) (Dade Behring) and a cartridge coated with collagen (fibrillar type 1 equine tendon) and ADP. Administration of Desmopressin—Desmopressin was diluted in sterile saline and infused over 30 min via the tail vein using a 10-ml syringe, "Flowline" Springfusor™ syringe driver, and Springfusor flow control tubing (Pacific Medical Supplies, Victoria, Australia) and a 30-guage 0.5-inch needle. For the time course experiment, the concentration of desmopressin was adjusted to deliver 3 μg/kg at a fixed volume of 250 μl over 30 min to six TSP-1+/+ or TSP-1-/- mice. Blood was sampled 1 h before desmopressin infusion and at 1 and 6 h postinfusion. The first two collections were by saphenous vein bleeds, and the third was by cardiac puncture. In separate experiments, desmopressin was infused over 30 min to three TSP-1+/+ or TSP-1-/- mice, and blood was collected by cardiac puncture at 1 h in one study and 6 h in another. Statistics—Comparative data are presented as means ± S.D. Statistical significance was calculated with Student's t test for all analyses. The average multimer size of VWF in plasma and platelet samples was estimated using two different measures. Samples were resolved on 1% agarose gel electrophoresis and the VWF was detected using 125I-labeled anti-human VWF polyclonal antibodies. The densities of the resulting multimer patterns were quantified and expressed as band intensity as a function of size. To compensate for small variations in protein loading and to better compare average VWF multimer size in a given experiment, the optical densities of the individual lanes were normalized so that the total density for each lane was the same. The CBA was also measured and expressed relative to the total VWF concentration (VWFAg) in the sample. The CBA/VWFAg ratio correlates with the average molecular weight of the intermediate and high VWF multimer forms for a given concentration of VWF (27Favaloro E.J. Facey D. Grispo L. Am. J. Clin. Pathol. 1995; 104: 264-271Crossref PubMed Scopus (66) Google Scholar). The overall error for the CBA/VWFAg ratio was calculated by adding the relative errors (one S.D.) for the individual CBA and VWFAg measures. TSP-1 influences the average multimer size of only the very large VWF multimers (10Xie L. Chesterman C.N. Hogg P.J. J. Exp. Med. 2001; 193: 1341-1349Crossref PubMed Scopus (112) Google Scholar, 17Xie L. Chesterman C.N. Hogg P.J. Thromb. Haemostasis. 2000; 84: 506-513Crossref PubMed Scopus (40) Google Scholar), which are difficult to resolve by gel electrophoresis or other means. Proteolysis of VWF by ADAMTS13, on the other hand, is readily apparent using this technique. The difficulty in resolving the very large VWF multimers can lead to uncertainty in the interpretation of the electrophoresis experiments. Importantly, however, the relative average VWF multimer size measured by gel electrophoresis in our study always correlated with the CBA/VWFAg ratio in a given experiment. Plasma VWF Multimer Size Was Smaller in TSP-1 Null than in Wild Type Mice—To evaluate the contribution of TSP-1 to the control of plasma VWF multimer size, we measured VWF multimer size in the plasmas of TSP-1+/+ and TSP-1-/- mice. Based on the in vitro evidence that TSP-1 reduces VWF multimer size (10Xie L. Chesterman C.N. Hogg P.J. J. Exp. Med. 2001; 193: 1341-1349Crossref PubMed Scopus (112) Google Scholar), we anticipated that in its absence the multimers might be larger in the TSP-1-/- mice. To the contrary, in both individual and pooled murine plasma, VWF multimer size was significantly smaller in TSP-1-/- mouse plasma. The ultralarge VWF multimers in the plasmas of individual mice were larger in TSP-1+/+ mice than in TSP-1-/- mice (Fig. 1A). This difference was confirmed by densitometry (Fig. 1B). The CBA to VWFAg ratios of the TSP-1+/+ cohort plasmas was also significantly higher than for the TSP-1-/- cohort (Fig. 1C). To allow for individual variations in multimer size between mice within a cohort, VWF multimer size was also measured in pooled plasma. Blood was collected by cardiac puncture from 12 (six male and six female) TSP-1+/+ or TSP-1-/- mice and an equal volume of blood from each mouse was mixed to prepare pooled plasma. This was repeated using three other groups of mice. The VWF multimer size in the pooled plasmas of the TSP-1-/- mice was significantly smaller than in the TSP-1+/+ mice in each group (Fig. 1, D and E). The Multimer Size of Endothelium-derived VWF Was Reduced More Rapidly in TSP-1 Null than in Wild Type Mice—Desmopressin is a synthetic analog of arginine vasopressin. The infusion of desmopressin to mice (28Sweeney J.D. Novak E.K. Reddington M. Takeuchi K.H. Swank R.T. Blood. 1990; 76: 2258-2265Crossref PubMed Google Scholar) and humans (29Richardson D.W. Robinson A.G. Ann. Intern. Med. 1985; 103: 228-239Crossref PubMed Google Scholar) results in a rapid increase in plasma VWF, which persists for 6 h or more. Desmopressin is thought to act via vasopressin receptors on endothelial cells to stimulate the release of VWF from endogenous stores. The increase in plasma VWF is accompanied by an increase in the concentration of ultralarge VWF multimers in the circulation (30Batlle J. Lopez-Fernandez M.F. Lopez-Borrasca A. Lopez-Berges C. Dent J.A. Berkowitz S.D. Ruggeri Z.M. Zimmerman T.S. Blood. 1987; 70: 173-176Crossref PubMed Google Scholar). The intravenous infusion of desmopressin to TSP-1+/+ and TSP-1-/- mice resulted in an increase in plasma VWF levels, which persisted at 6 h. The increase in plasma VWF concentration in TSP-1+/+ mice (Fig. 2A), however, was accompanied by a greater relative increase in the ultralarge VWF multimers than was observed in TSP-1-/- mice (Fig. 2B). The average multimer size of plasma VWF was larger in TSP-1+/+ than TSP-1-/- mice at both 1 h (Fig. 2C) and 6 h (Fig. 2D) after desmopressin treatment. This result indicated that ultralarge VWF multimers released from stimulated endothelial cells in vivo are more efficiently processed in the absence of plasma TSP-1. TSP-1 Bound to the A3 Domain of VWF—The interaction of TSP-1 and TSP-1 fragments with the A3 domain of VWF was measured using a competitive binding technique. In this technique, A3 immobilized on polystyrene was used as a probe to monitor the solution phase binding of TSP-1 or E3CaG-1 (which contains the Ca2+-binding repeats and C-terminal sequence and harbors the VWF reductase activity) to A3. Interaction of ligands with protein acceptors immobilized on plastic often introduces artifacts due to varying degrees of denaturation of the plastic-bound protein. This was probably the cause of the lack of saturable binding of TSP-1 (Fig. 3B) and E3CaG-1 (Fig. 3D) to immobilized VWF A3. Soluble A3 competed for the binding of TSP-1 (Fig. 3C) and E3CaG-1 (Fig. 3E) to immobilized A3, however, which implied that the interaction was specific. Apparent dissociation constants for binding of TSP-1 or E3CaG-1 to soluble A3 of ∼3 and ∼6 μm, respectively, were estimated from the competition experiments. As expected, soluble E3CaG-1, but not the CP123-1 fragment (which contains the procollagen-like module and three properdin-like or type 1 modules), competed with TSP-1 for binding to immobilized A3 (Fig. 3, E and F). These results indicate that TSP-1, like ADAMTS13 (31Dong J.F. Moake J.L. Bernardo A. Fujikawa K. Ball C. Nolasco L. Lopez J.A. Cruz M.A. J. Biol. Chem. 2003; 278: 29633-29639Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), interacts with the A3 domain of VWF. The Average Multimer Size of the VWF in Human Platelets Is Smaller than in Murine Platelets—Variation in the pattern and processing of VWF multimer size in human and murine platelets was evaluated by lysing platelets in the absence or presence of the TSP-1 inhibitors, EDTA and NEM. There was a partial reduction in multimer size of both human and murine platelet VWF upon lysis in the absence of the TSP-1 inhibitors (Fig. 4). Notably, the VWF multimers in human platelets are significantly smaller than in C57BL/6 murine platelets. Platelet VWF Multimer Size Was Reduced upon Lysis of Wild Type but Not TSP-1 Null Platelets—To study the role of TSP-1 in regulating platelet VWF multimer size, we prepared platelet lysates from TSP-1+/+ and TSP-1-/- mice. The platelet lysates were prepared in the presence of a range of protease inhibitors, including phenanthroline that inactivates ADAMTS13 (8Tsai H.M. Blood. 1996; 87: 4235-4244Crossref PubMed Google Scholar) and either with or without the TSP-1 inhibitors, EDTA and NEM. The VWF reductase activity of TSP-1 is inactivated by EDTA, which depletes the C-terminal Ca2+-binding repeats of this ion, and by NEM, which alkylates the critical thiol at Cys974. The average VWF multimer size was partially reduced in TSP-1+/+ but not in TSP-1-/- platelets when lysed without the TSP-1 inhibitors (Fig. 5). The reduction in multimer size was more obvious when measured by collagen avidity, which preferentially