Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network.
Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.
From the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, 6229 EV, Maastricht, The Netherlands, Synapse BV, 6229 EV, Maastricht, The Netherlands, adam.miszta@thrombin.com.
Adam
A
Miszta
From the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, 6229 EV, Maastricht, The Netherlands, Synapse BV, 6229 EV, Maastricht, The Netherlands.
Leonie
L
Pelkmans
From the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, 6229 EV, Maastricht, The Netherlands, Synapse BV, 6229 EV, Maastricht, The Netherlands.
Theo
T
Lindhout
From the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, 6229 EV, Maastricht, The Netherlands.
Ganeshram
G
Krishnamoorthy
Clinical Chemistry and Hematology, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
Philip G
PG
de Groot
From the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, 6229 EV, Maastricht, The Netherlands, Synapse BV, 6229 EV, Maastricht, The Netherlands.
Coenraad H
CH
Hemker
From the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, 6229 EV, Maastricht, The Netherlands.
Johan W M
JW
Heemskerk
From the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, 6229 EV, Maastricht, The Netherlands, Synapse BV, 6229 EV, Maastricht, The Netherlands.
Hilde
H
Kelchtermans
From the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, 6229 EV, Maastricht, The Netherlands, Synapse BV, 6229 EV, Maastricht, The Netherlands.
Bas
B
de Laat
D000595:Amino Acid Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000502:physiology; D005337:Fibrin / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D010974:Platelet Aggregation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D013917:Thrombin / Q000737:chemistry*; D014841:von Willebrand Factor / Q000737:chemistry*
D014841:von Willebrand Factor; D005337:Fibrin; D013917:Thrombin
Arvin; Ellipsometry; Fibrin; Fibrinogen; Platelet; Platelet Glycoprotein Ib; Surface Plasmon Resonance (SPR); von Willebrand Factor