1. Activation of Human Platelets by the Membrane-Expressed A1 Domain of von Willebrand Factor
by Jan Schulte am Esch, Miguel A. Cruz, Jonathan B. Siegel, Josef Anrather, and Simon C. Robson
Blood
Volume 90(11):
December 1, 1997
©1997 by American Society of Hematology

2. Outline structure of the FLAG-tagged GPI-anchored vWF–A1-domain fusion protein expressed on COS-7 cells.
Outline structure of the FLAG-tagged GPI-anchored vWF–A1-domain fusion protein expressed on COS-7 cells. The secretion sequence of preprotrypsin, including the FLAG-epitope, was cloned as an oligonucleotide into the pcDNA3 vector. The human and porcine vWF sequence (aa ) cloned from porcine and human endothelial cell RNA by RT-PCR and a further truncated fragment (aa 475*-709*) generated by PCR from human A1 (aa ) were separately ligated into the vector. Finally, the C-terminal DAF-derived GPI-linker sequence was inserted into the construct.
Jan Schulte am Esch II et al. Blood 1997;90:
©1997 by American Society of Hematology

3. Jan Schulte am Esch II et al. Blood 1997;90:4425-4437
©1997 by American Society of Hematology

4. Cytofluorometric analysis of vWF–A1-domain expressed on COS-7 cells.
Cytofluorometric analysis of vWF–A1-domain expressed on COS-7 cells. Transfected cells were first incubated with the monoclonal mouse antibodies anti-FLAG (A and B), LJ-RG-46, specifically detecting human A1-domain (C), and anti–c-myc (D) as a nonspecific control followed by incubation with a FITC-conjugated anti-mouse antibody for 20 minutes at 4°C. At least 20,000 cells were counted each experiment.
Jan Schulte am Esch II et al. Blood 1997;90:
©1997 by American Society of Hematology

5. Jan Schulte am Esch II et al. Blood 1997;90:4425-4437
©1997 by American Society of Hematology

6. Jan Schulte am Esch II et al. Blood 1997;90:4425-4437
©1997 by American Society of Hematology

7. Jan Schulte am Esch II et al. Blood 1997;90:4425-4437
©1997 by American Society of Hematology

8. Jan Schulte am Esch II et al. Blood 1997;90:4425-4437
©1997 by American Society of Hematology

9. Jan Schulte am Esch II et al. Blood 1997;90:4425-4437
©1997 by American Society of Hematology

10. Platelet aggregation and Ca++ influx.
Platelet aggregation and Ca++ influx. Washed platelets were prepared as described under Materials and Methods. For intracellular Ca++ uptake (Δ[Ca2+]i) detection, platelets were loaded with the photoprotein aequorin. MgCl2 (1.0 mmol/L), CaCl2 (1.0 mmol/L), and fibrinogen (final concentration: 800 μg/mL) were added before the experiments. Aggregation and luminescence following incubation with transfected COS-7 cells were recorded simultaneously. Each box displays one representative experiment for COS-7 to platelet ratios of 1:280. The upper (+)-tagged lines show human platelet aggregation as percentage of light transmission. The lower (×)-tagged lines give the simultaneously recorded change in luminescence due to platelet Δ[Ca2+]i .
Jan Schulte am Esch II et al. Blood 1997;90:
©1997 by American Society of Hematology

11. Jan Schulte am Esch II et al. Blood 1997;90:4425-4437
©1997 by American Society of Hematology

12. Jan Schulte am Esch II et al. Blood 1997;90:4425-4437
©1997 by American Society of Hematology

13. Effect of ristocetin on aggregation of HWP after adding porcine and human vWF–A1-domain transfected COS-7 cells.
Effect of ristocetin on aggregation of HWP after adding porcine and human vWF–A1-domain transfected COS-7 cells. HWP were prepared as described in Materials and Methods. The extent of aggregation is represented by the percentage of the maximal light transmission 10 minutes following addition of porcine (P) and human (H) vWF–A1-domain expressing COS-7 cells at low ratios (1:1,200 cells/platelets). Incubation of HWP with ristocetin (R) (0.32 mg/mL) alone or in combination with empty vector–transfected COS-7 cells (E) excluded significant human vWF contamination in platelet preparations. Specificity for A1-domain–GPIb interaction was further proven by incubating platelets with 6D1, a specific antibody known to block GPIb-mediated aggregation by vWF.
Jan Schulte am Esch II et al. Blood 1997;90:
©1997 by American Society of Hematology

14. Bone marrow erythroblasts from a patient with congenital dyserythropoietic anemia type I: chromatin bridges between two adjacent nuclear lobes (arrow-head), spongy appearance of the chromatin (stars), and widening of nuclear pores with nucleus invasion by t...
Bone marrow erythroblasts from a patient with congenital dyserythropoietic anemia type I: chromatin bridges between two adjacent nuclear lobes (arrow-head), spongy appearance of the chromatin (stars), and widening of nuclear pores with nucleus invasion by the cytoplasm (arrow) are characteristic ultrastructural features of the disease. Original magnification × 10,400. (Courtesy of Elisabeth M. Cramer and Josette Guichard, INSERM U.91, Hopital Henri Mondor, Creteil, France.)‏
Jan Schulte am Esch II et al. Blood 1997;90:
©1997 by American Society of Hematology