von Willebrand factor binds to native collagen VI primarily via its A1 domain.

Collagen VI is abundant in the arterial subendothelium. To investigate its mechanism of interaction with von Willebrand factor (vWF), collagen VI was isolated from human placenta and from the extracellular matrix of the human lung fibroblast cell line MRC-5. Purified vWF bound to non-digested collagen VI with moderately high affinity (EC50 approximately 5 nM) and could be inhibited by the Hirudo medicinalis collagen inhibitor calin. The anti-(human vWF A1 domain) monoclonal antibody (AJvW-2), as well as aurin tricarboxylic acid (ATA), at concentrations that saturate the vWF A1 domain, also inhibited this binding. In contrast, the monoclonal anti-(human vWF A3 domain) antibody (82D6A3) inhibited vWF binding to collagens I, III and IV, but had no effect on vWF binding to collagen VI. Likewise, vWF binding to collagen VI was not inhibited by the recombinant vWF domain D4. Polyclonal anti-(collagen VI) antibodies, specifically neutralizing the binding of vWF to collagen VI, confirmed that in the intact endothelial cell extracellular matrix, collagen VI was accessible for interaction with vWF. This binding was only marginally affected by 82D6A3 but was dose-dependently inhibited by AJvW-2, ATA and the A1 domain analogue VCL (recombinant A1 domain of vWF), with IC50 values comparable to those found for the inhibition of vWF binding to isolated collagen VI. The weak interaction of isolated human platelets with collagen VI was mediated via the platelet collagen receptor (GPIa/IIa) and was competitively inhibited by vWF but not by VCL, suggesting that vWF and GPIa/IIa bind to neighbouring but distinct sites on collagen VI. We conclude that vWF binds to collagen VI primarily via its A1 domain, which distinguishes it from the vWF A3 domain-mediated binding to fibrillar collagens.

Prolonged inhibition of acute arterial thrombosis by high dosing of a monoclonal anti-platelet glycoprotein IIb/IIIa antibody in a baboon model.

The in vivo activity of MA-16N7C2, the first monoclonal antibody that contains an echistatin-like RGD-sequence and inhibits platelet glycoprotein (GP)IIb/IIIa function, was determined in baboons. A dose-finding study assessing haemostatic variables such as bleeding time and ex vivo platelet aggregation showed that doses of as low as 0.2-0.3 mg/kg resulted in a pronounced effect. The effects were dose-dependent and lasted for several days, implying that MA-16N7C2 is a potent and long-acting GPIIb/IIIa inhibitor. Following the initial studies, the antithrombotic effect of 0.1 and 0.3 mg/kg of the antibody, given as a bolus, was determined in a baboon model of platelet-dependent, arterial-type thrombus formation. In these studies, a thrombogenic device consisting of Dacron vascular graft material was inserted as extension segments into a permanent arteriovenous shunt. The results confirmed the potent and long-lasting antithrombotic effect of MA-16N7C2. Surprisingly, the antithrombotic effect was stronger 48 h after a dose of 0.3 mg/kg administration than on the day of treatment with 0.1 mg/kg, despite the fact that comparable numbers of GPIIb/IIIa receptors were occupied on resting platelets. We postulate that with the high dose of MA-16N7C2 and after an extended period, occupied GPIIb/IIIa may be internalised by the platelets. Upon platelet activation, these receptors become reexposed but are unable to participate in thrombus formation. This is in contrast to unoccupied internal GPIIb/IIIa receptors early after a low dose of MA-16N7C2.

Promotion of binding of von Willebrand factor to platelet glycoprotein Ib by dimers of ristocetin.

In the absence of high shear forces, the in vitro binding of human von Willebrand factor (vWF) to its platelet receptor glycoprotein Ib (GPIb) can be promoted by two well-characterized mediators, botrocetin and ristocetin. Using purified vWF and GPIb, we have investigated the mechanisms by which ristocetin mediates this binding. Specific binding of vWF monomers to GPIb occurred with a 1:1 stoichiometry, but high-affinity binding required the participation of two ristocetin dimers. Binding was strongly dependent on pH and inhibited by low poly-L-lysine concentrations, indicating ristocetin-dependent charge neutralization during the interaction. With increasing ristocetin concentrations, vWF binding depended progressively less on the involvement of its A1 loop, which is compatible with a model in which the two ristocetin dimers bridge the vWF-GPIb complex on secondary sites. In agreement with this model, the ristocetin-dimer-promoted stabilization of vWF on GPIb was abolished by low concentrations of poly(Pro-Gly-Pro), which is known to complex ristocetin dimers. Mechanistic analysis of the inhibition of vWF binding by the recombinant vWF fragment Leu504-Ser728 (VCL), which covers the entire A1 loop, revealed an affinity of VCL for GPIb comparable with that of the botrocetin-vWF complex for GPIb, and identified a specific but 20-fold lower affinity of VCL in the presence of ristocetin. The proline-rich peptides flanking the vWF A1 loop, Cys474-Val489 and Leu694-Asp709, inhibited vWF binding semispecifically by competitively interfering with the formation of the GPIb-vWF complex rather than by complexation of free ristocetin dimers. In conclusion, ristocetin-promoted binding of vWF to its GPIb receptor results from charge neutralization and interactions involving proline residues in the vicinity of the natural interaction sites present on both GPIb and the A1 domain of vWF.