von Willebrand factor contained in a high purity FVIII concentrate (Fanhdi) binds to platelet glycoproteins and supports platelet adhesion to subendothelium under flow conditions.
OBJECTIVE
There is evidence suggesting that von Willebrand factor (VWF) from high purity factor VIII concentrates could be of clinical use in the management of patients suffering from VWD. We analyzed structural and functional characteristics of VWF present in a high purity factor VIII concentrate VWFHPC (Fanhdi). The multimeric structure, the ability to bind to platelet GP Ib/IX or GP IIb/IIIa, and the capacity of VWFHPC to promote platelet adhesion on injured vessels were investigated and compared with that present in standard plasma cryoprecipitates [VWFCRYO].


METHODS
Binding studies were carried out by incubating radiolabeled VWF and washed platelets, which were activated with either ristocetin (1 mg/mL; for GP Ib/IX), or thrombin (2.5 U/mL; for GP IIb/IIIa). Platelet adhesion was assessed in a perfusion system (shear rate = 800 s-1, 10 min) in which the source of VWF was added (at 0.4 or 0.8 U/mL VWF:Ag) to washed platelets and red cells suspended in a human albumin solution. The deposition of platelets onto the perfused subendothelial surface was morphometrically evaluated and expressed as percentage of surface coverage (%SC).


RESULTS
The VWFHPC (152 Units VWF:RCof/mg protein; VWF:RCof/VWF:Ag = 0.97), lacked only a small proportion of high-molecular-weight multimers present in VWFCRYO. Binding affinities (Kd values, nM) of VWFHPC were similar to those of VWFCRYO (5.3 +/- 0.86 vs 5.2 +/- 0.95, for GP Ib/IX; and 11.6 +/- 2.7 vs 15.4 +/- 1.7 for GPIIb-IIIa). A slightly, though not significantly, higher binding capacity for these receptors (Bmax values, molecules/pit) was obtained for VWFHPC. The %SC in perfusions in the presence of albumin was < 10%. Addition of VWFHPC or VWFCRYO significantly increased the %SC, with values of 27.1 +/- 4.9 and 17.5 +/- 2.8%, respectively with 0.4 U/mL (p < 0.004 and p < 0.02 vs albumin); and 30.8 +/- 4.9% and 20.03 +/- 4.1%, respectively, at 0.8 U/mL (p < 0.001 and p < 0.02 vs albumin).


CONCLUSIONS
Our data show that VWF present in the high purity FVIII concentrate Fanhdi retains the functional capacity to bind to GPs Ib/IX and IIb/IIIa and to promote platelet adhesion onto exposed subendothelium.
Rivera
J
J
Hematology Unit, Hospital General Universitario, Murcia, Spain.
Escolar
G
G
Casamiquela
R
R
Bravo
M I
MI
Jorquera
J I
JI
Castillo
R
R
Ordinas
A
A
Vicente
V
V
D000418:Albumins / Q000378:metabolism; D001667:Binding, Competitive; D011232:Chemical Precipitation; D003080:Cold Temperature; D005169:Factor VIII / Q000737:chemistry*; D006801:Humans; D008970:Molecular Weight; D010477:Perfusion; D010973:Platelet Adhesiveness / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012212:Rheology; D012310:Ristocetin / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014841:von Willebrand Factor / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000418:Albumins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; C032193:glycoprotein receptor GPIb-IX; D014841:von Willebrand Factor; C028254:von Willebrand factor receptor; D012310:Ristocetin; D005169:Factor VIII