Effect of von Willebrand Factor on the Pharmacokinetics of Recombinant Human Platelet Glycoprotein Ibα-Immunoglobulin G1 Chimeric Proteins

Wang, Qin; Shorten, Douglas; Xu, Xin; Shaw, Gray D.; Schaub, Robert G.; Shea, Christopher R.; Brooks, Jonathan; Sako, Dianne; Wiswall, Erin; Xu, Jin; Szklut, Pamela; Patel, Vikram S.

doi:10.1007/s11095-006-9018-1

Cited by 3 publications

(6 citation statements)

References 27 publications

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“…These results suggest that GPIb␣-Ig/2V is both a cofactor for AD-AMTS13 because it binds VWF domain A1, 12 and an antagonist of shear-induced VWF cleavage because it inhibits VWF binding to platelets. 21 These results demonstrate that platelets increase the sheardependent cleavage of VWF by ADAMTS13 using purified proteins. To establish that a similar effect occurs under more physiologic conditions, similar studies were performed in plateletrich and platelet-poor plasma anticoagulated with heparin, prostacyclin and D-Phe-Pro-Arg-chloromethane ( Figure 1D).…”

mentioning

confidence: 64%

“…28,29 These mutations increase the affinity of binding to VWF, and GPIb␣-Ig/2V binds immobilized VWF with an IC 50 of 0.5 g/mL. 21 At the concentrations of VWF and GPIb␣-Ig/2V used in these experiments (30 g/mL each), almost all GPIb␣-Ig/2V binding sites on VWF will be occupied. In the absence of platelets, GPIb␣-Ig/2V substantially increased the cleavage of VWF ( Figure 1C Figure 1C lane 8).…”

Section: Resultsmentioning

confidence: 98%

“…21 This construct contains 2 mutations that cause platelet-type or pseudo-von Willebrand disease, G233V and M239V. 28,29 These mutations increase the affinity of binding to VWF, and GPIb␣-Ig/2V binds immobilized VWF with an IC 50 of 0.5 g/mL.…”

Section: Resultsmentioning

confidence: 99%

“…Complete reaction mixtures were preincubated at 37°C for 10 minutes and then subjected to a constant fluid shear stress for various times. In some experiments, platelets were preincubated for 30 minutes with 30 g/mL of monoclonal anti-human GPIb␣ antibody 6D1 20 provided by Dr Barry Coller (Rockefeller University, New York, NY), a recombinant chimeric protein consisting of human GPIb␣ residues 1-290 fused to the Fc region of human IgG1 (GPIb␣-Ig/2V), 21 or mouse IgG1 (BioLegend, San Diego, CA). Reactions were stopped by mixing 15-L samples with an equal volume of Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA).…”

Section: Shear-induced Proteolytic Cleavage Of Vwfmentioning

confidence: 99%

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Platelet-VWF complexes are preferred substrates of ADAMTS13 under fluid shear stress

Shim

Anderson

Tuley

et al. 2008

Blood

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144

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Introductionvon Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays an essential role in tethering platelets at the site of vascular injury. VWF is synthesized in endothelial cells, where a portion is stored in granules called Weibel-Palade bodies as "unusually large" or "ultralarge" multimers (ULVWF) and secreted upon endothelial stimulation. 1,2 Secreted ULVWF multimers bind platelets with relatively high affinity and are thought to be prothrombotic. ULVWF is cleaved into smaller and less dangerous multimers by the metalloprotease ADAMTS13, a member of the A Disintegrin And Metalloprotease with ThromboSpondin type I repeat family. [3][4][5] Inherited or acquired deficiency of ADAMTS13 causes life-threatening microvascular thrombosis that is characteristic of thrombotic thrombocytopenic purpura. 4,6,7 Conversely, mutations in von Willebrand disease type 2A cause bleeding by increasing the cleavage of VWF by ADAMTS13 and impairing platelet adhesion. [8][9][10] Therefore, normal hemostasis depends on the precise regulation of VWF proteolysis.ADAMTS13 cleaves the Tyr 1605 -Met 1606 bond in the A2 domain of VWF, but this bond is buried and relatively inaccessible until the A2 domain is unfolded, presumably by tensile force in vivo. 10,11 The shear stress required to apply this force will vary depending on whether VWF is immobilized at the vessel wall or moving with the flowing blood, and whether platelets are bound to it. The rate of VWF cleavage also can be modulated by cofactors that bind to the A1 domain, including platelet GPIb and heparin. 12 Thus, ADAMTS13 is presented with VWF multimers in plasma or on endothelial cell surfaces that vary in their susceptibility to cleavage, with or without attached platelets.The relevance of each of these potential substrates to the catabolism of VWF is unknown. Several studies suggest that VWF strings on endothelial cells must be cleaved to inhibit thrombus growth, 13,14 but the role of proteolysis in the fluid phase has not been established. Therefore, the cleavage of VWF by ADAMTS13 was assessed in a cone-plate viscometer to minimize the contribution of surface interactions. The results indicate that proteolysis of fluid phase VWF-platelet complexes is likely to determine the steady state size distribution of circulating VWF multimers in vivo. Methods Recombinant ADAMTS13Full-length human ADAMTS13 with a C-terminal V5 tag was expressed in TRex 293 cells (Invitrogen) as described previously 15 and partially purified by anion exchange chromatography. In brief, conditioned medium containing recombinant ADAMTS13 was supplemented with proteinase inhibitors (0.1 mol/L D-Phe-Pro-Arg-chloromethane and 144 mol/L phenylmethylsulfonyl fluoride and applied to tandem columns of HiTrap Q Sepharose (2 ϫ 5 mL; GE Healthcare, Chalfont St Giles, United Kingdom). The columns were washed with 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and developed with a linear gradient of 0 to 50 mM CaCl 2 in 20 mM Tris-HCl, pH 8.0, and 100 mM NaCl. Fractions containing ADAMTS13 were combined,...

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mentioning

confidence: 64%

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Shear-induced Proteolytic Cleavage Of Vwfmentioning

confidence: 99%

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Platelet-VWF complexes are preferred substrates of ADAMTS13 under fluid shear stress

Shim

Anderson

Tuley

et al. 2008

Blood

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149

144

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“…In addition, increased serum levels of vWF have been reported in CRC patients and have been related to the progression of tumor and dissemination of malignant cells. 22,23 Remarkably, vWF was absent or only weakly expressed in all of the eight TEC cultures, but was highly expressed in five of the corresponding NEC cultures. NEC of three patients displayed only weak levels of vWF.…”

Section: Discussionmentioning

confidence: 95%

Endothelial cells of human colorectal cancer and healthy colon reveal phenotypic differences in culture

Schellerer

Croner

Weinländer

et al. 2007

Laboratory Investigation

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Antiangiogenic drugs have been used successfully for the treatment of colorectal cancer (CRC). Viable tumor endothelial cells (TEC) and normal endothelial cells (NEC) of uninvolved colon tissue of the same patient have not been available to optimize treatment strategies in vitro. Therefore, our target was to establish a protocol for the isolation of TEC and NEC. These cells were isolated with very high purity via magnetic cell sorting of tissue samples obtained from CRC and healthy colon of eight patients. TEC and NEC expressed CD31, CD105, VE-cadherin, VCAM-1, ICAM-1 and E-selectin, formed capillaries in basal membrane extract and were able to take up acetylated low-density lipoprotein. They were negative for podoplanin, CD45, CD68 and cytokeratin-20 indicating blood vessel endothelial lineage. Intense staining of von Willebrand factor (vWF) was observed in five of eight NEC cultures, whereas vWF was absent or only slightly expressed in all TEC cultures in vitro. Low intracellular concentration of vWF was also detected in TEC and NEC at the tissue level. This demonstrated that differences exhibited by TEC and NEC in vivo are stably perpetuated in culture. The isolated cultures may provide a useful in vitro model to elucidate epigenetic effects on angiogenesis in cancer and to optimize antiangiogenic therapy.

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Identification of Peptide Antagonists to Glycoprotein Ibα That Selectively Inhibit von Willebrand Factor Dependent Platelet Aggregation

Benard¹,

Smith²,

Cunningham³

et al. 2008

Biochemistry

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GPIbalpha is an integral membrane protein of the GPIb-IX-V complex found on the platelet surface that interacts with the A1 domain of von Willebrand factor (vWF-A1). The interaction of GPIbalpha with vWF-A1 under conditions of high shear stress is the first step in platelet-driven thrombus formation. Phage display was used to identify peptide antagonists of the GPIbalpha-vWF-A1 interaction. Two nine amino acid cysteine-constrained phage display libraries were screened against GPIbalpha revealing peptides that formed a consensus sequence. A peptide with sequence most representative of the consensus, designated PS-4, was used as the basis for an optimized library. The optimized selection identified additional GPIbalpha binding peptides with sequences nearly identical to the parent peptide. Surface plasmon resonance of the PS-4 parent and two optimized synthetic peptides, OS-1 and OS-2, determined their equilibrium dissociation GPIbalpha binding constants ( K Ds) of 64, 0.74, and 31 nM, respectively. Isothermal calorimetry corroborated the K D of peptide PS-4 with a resulting affinity value of 68 nM. An ELISA demonstrated that peptides PS-4, OS-1, and OS-2 competitively inhibited the interaction between the vWF-A1 domain and GPIbalpha-Fc in a concentration-dependent manner. All three peptides inhibited GPIbalpha-vWF-mediated platelet aggregation induced under high shear conditions using the platelet function analyzer (PFA-100) with full blockade observed at 150 nM for OS-1. In addition, OS-1 blocked ristocetin-induced platelet agglutination of human platelets in plasma with no influence on platelet aggregation induced by several agonists of alternative platelet aggregation pathways, demonstrating that this peptide specifically disrupted the GPIbalpha-vWF-A1 interaction.

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Effect of von Willebrand Factor on the Pharmacokinetics of Recombinant Human Platelet Glycoprotein Ibα-Immunoglobulin G1 Chimeric Proteins

Abstract: The difference in disposition for valine substituted forms of GPIbalpha-Ig in laboratory animals are likely affected by their enhanced binding affinity for circulating vWF.

Cited by 3 publications

References 27 publications

Platelet-VWF complexes are preferred substrates of ADAMTS13 under fluid shear stress

Platelet-VWF complexes are preferred substrates of ADAMTS13 under fluid shear stress

Endothelial cells of human colorectal cancer and healthy colon reveal phenotypic differences in culture

Identification of Peptide Antagonists to Glycoprotein Ibα That Selectively Inhibit von Willebrand Factor Dependent Platelet Aggregation

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