scholarly journals Characterization of 25 monoclonal antibodies to factor VIII-von Willebrand factor: relationship between ristocetin-induced platelet aggregation and platelet adherence to subendothelium

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1408-1415 ◽  
Author(s):  
HV Stel ◽  
KS Sakariassen ◽  
BJ Scholte ◽  
EC Veerman ◽  
TH van der Kwast ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Primary Hemostasis ◽  
Platelet Adherence ◽  
Physiologic Mechanism ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

Abstract We have studied the role of factor VIII-von Willebrand factor (FVIII- vWF) in both platelet adherence to subendothelium and ristocetin- induced platelet aggregation using monoclonal antibodies to human FVIII- vWF. Twenty-five monoclonal antibodies were obtained, two of which were directed to the factor VIII moiety of FVIII-vWF; one of these two completely inhibited the procoagulant activity (FVIII:C). The remaining 23 monoclonal antibodies were directed to the von Willebrand factor moiety of FVIII-vWF. The ability of the latter monoclonal antibodies to inhibit platelet adherence to arterial subendothelium was investigated with a perfusion model. According to the number of platelets adhering to the subendothelium, three groups of monoclonal antibodies could be discerned: (A) antibodies not affecting platelet adherence; (B) antibodies that inhibited platelet adherence to the level as observed when von Willebrand's disease plasma was tested; and (C) antibodies that completely inhibited both platelet adherence to subendothelium and ristocetin-induced platelet aggregation. The two antibodies present in group C competed for the same or closely related epitope(s) present on FVIII-vWF. These results demonstrate that a domain is present on the FVIII-vWF molecule that is associated both with ristocetin-induced aggregation and with the ability of FVIII-vWF to support platelet adherence to the subendothelium. Based on these observations, it is concluded that ristocetin-induced binding of FVIII-vWF to platelets reflects, at least in part, a physiologic mechanism regulating the function of FVIII-vWF in primary hemostasis.

scholarly journals Characterization of 25 monoclonal antibodies to factor VIII-von Willebrand factor: relationship between ristocetin-induced platelet aggregation and platelet adherence to subendothelium

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1408-1415 ◽  
Author(s):  
HV Stel ◽  
KS Sakariassen ◽  
BJ Scholte ◽  
EC Veerman ◽  
TH van der Kwast ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Primary Hemostasis ◽  
Platelet Adherence ◽  
Physiologic Mechanism ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

We have studied the role of factor VIII-von Willebrand factor (FVIII- vWF) in both platelet adherence to subendothelium and ristocetin- induced platelet aggregation using monoclonal antibodies to human FVIII- vWF. Twenty-five monoclonal antibodies were obtained, two of which were directed to the factor VIII moiety of FVIII-vWF; one of these two completely inhibited the procoagulant activity (FVIII:C). The remaining 23 monoclonal antibodies were directed to the von Willebrand factor moiety of FVIII-vWF. The ability of the latter monoclonal antibodies to inhibit platelet adherence to arterial subendothelium was investigated with a perfusion model. According to the number of platelets adhering to the subendothelium, three groups of monoclonal antibodies could be discerned: (A) antibodies not affecting platelet adherence; (B) antibodies that inhibited platelet adherence to the level as observed when von Willebrand's disease plasma was tested; and (C) antibodies that completely inhibited both platelet adherence to subendothelium and ristocetin-induced platelet aggregation. The two antibodies present in group C competed for the same or closely related epitope(s) present on FVIII-vWF. These results demonstrate that a domain is present on the FVIII-vWF molecule that is associated both with ristocetin-induced aggregation and with the ability of FVIII-vWF to support platelet adherence to the subendothelium. Based on these observations, it is concluded that ristocetin-induced binding of FVIII-vWF to platelets reflects, at least in part, a physiologic mechanism regulating the function of FVIII-vWF in primary hemostasis.

Interactions of Purified Rat Factor VIII/von Willebrand Factor with Rat and Human Platelets – Effect of Albumin and Ristocetin

1984 ◽  
Vol 52 (01) ◽  
pp. 057-059 ◽  
Author(s):  
E Dejana ◽  
M Furlan ◽  
B Barbieri ◽  
M B Donati ◽  
E A Beck
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Rat Plasma ◽  
Human Platelets ◽  
High Concentrations ◽  
Low Sensitivity ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor ◽  
Rat Platelets

SummaryRat platelets do not respond to ristocetin in their own plasma nor do they aggregate in the presence of bovine or porcine factor VIII von Willebrand factor (F VIII R:WF) or human F VIII R:WF in presence of ristocetin. However, rat plasma supports ristocetin induced aggregation of washed human platelets. In this study we report on purification of rat F VIII R:WF from cryoprecipitate. Similarly to porcine or bovine material, purified rat F VIII R:WF induced aggregation of human washed fixed platelets. This effect was enhanced by addition of ristocetin and was not modified by addition of albumin. Rat washed platelets were aggregated by ristocetin in the presence of rat or human F VIII R:WF provided that high concentrations of ristocetin are added in a system essentially free of extraneous proteins. Increasing concentrations of albumin dramatically reduced the ability of ristocetin to aggregate rat platelets while human platelet aggregation by human or rat F VIII R:WF was only moderately affected.These studies show that rat F VIII R:WF can interact with rat and human platelets. The lack of response of rat platelets to ristocetin in their own plasma is most likely due to a low sensitivity of rat platelets to this drug and to an inhibitory activity of plasma proteins on this reaction.

scholarly journals Effect of monoclonal antibodies against von Willebrand factor and platelet glycoproteins IIb/IIIa on the platelet retention test

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 546-550 ◽  
Author(s):  
J McPherson ◽  
S Brownlea ◽  
MB Zucker
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Retention Test ◽  
Platelet Adhesion ◽  
Glass Beads ◽  
Two Stage ◽  
Second Stage ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The platelet retention test provides a measure of the number of platelets retained in a column of glass beads and is one of the few in vitro platelet function tests that is abnormal in von Willebrand's disease (vWd). In a two-stage test, 1 mL of blood (designated A) was passed through the column, followed by 5 mL of isotonic saline and then 5 mL of blood (B) in which platelet retention was measured. With normal blood as A and B, retention is very high in all 5 mL of blood B. In the first stage, platelets adhere to the glass beads; this requires fibrinogen but not von Willebrand factor (vWf). The platelet-platelet adhesion in the second stage requires vWf, is dependent on release of ADP, and fails to occur if thrombasthenic platelets are tested. Retention was normal when blood from a patient with afibrinogenemia was used as blood B. We have now used monoclonal antibodies to elucidate further the mechanism of platelet retention. Five antibodies to different epitopes on vWf essentially abolished retention in the one- stage test and in the second stage of the two-stage test, but had no effect on the first stage. Thus, the entire vWf molecule must be free of antibody to function in the platelet-platelet adhesion of the second stage of this test. Binding of the antigen-antibody complex to the platelet Fc receptor was not responsible, as Fab and F(ab')2 fragments of one of the antibodies were as effective as intact antibody, and as neither heat-aggregated IgG nor a polyclonal antibody to plasma factor IX inhibited retention. F(ab')2 fragments of 6D1, an antibody to platelet GP Ib that prevents binding of vWf to platelets, also inhibited the second phase of retention. An antibody that inhibits binding of fibrinogen and vWf to GP IIb/IIIa (LJ-CP8) inhibited both the first and second stages of retention, whereas LJ-P5, an antibody that inhibits only the binding of vWf to GP IIb/IIIa, caused slight inhibition of retention when normal or afibrinogenemic blood was used as blood B and was reported to cause only partial inhibition of ADP- induced platelet aggregation in this afibrinogenemic patient. The results suggest that vWf is altered during rapid passage of blood through the glass-bead column so that it attaches to GP Ib, exposing GP IIb/IIIa, which then binds the altered vWf or fibrinogen, either of which can induce platelet aggregation (platelet-platelet adhesion) and thus retention in the column.

Structure and function of the von Willebrand factor A1 domain: analysis with monoclonal antibodies reveals distinct binding sites involved in recognition of the platelet membrane glycoprotein Ib-IX-V complex and ristocetin-dependent activation

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 164-172 ◽  
Author(s):  
Mariagrazia De Luca ◽  
David A. Facey ◽  
Emmanuel J. Favaloro ◽  
Mark S. Hertzberg ◽  
James C. Whisstock ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Membrane ◽  
Membrane Glycoprotein ◽  
Platelet Membrane Glycoprotein ◽  
And Function ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Binding of the adhesive glycoprotein, von Willebrand factor (vWf), to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates platelet adhesion and aggregation at high shear stress in hemostasis and thrombosis. In this study, the GP Ib-IX-V binding site within the vWf A1 domain was analyzed using a panel of murine monoclonal antibodies raised against a 39/34-kd vWf fragment (Leu-480/Val-481–Gly-718) encompassing the A1 domain. One antibody, 6G1, strongly inhibited ristocetin-dependent vWf binding to platelets, but had no effect on botrocetin- or jaracetin-dependent binding, or asialo-vWf–dependent platelet aggregation. The 6G1 epitope was mapped to Glu-700–Asp-709, confirming the importance of this region for modulation of vWf by ristocetin. Like ristocetin, 6G1 activated the vWf A1 domain, because it enhanced binding of the 39/34-kd fragment to platelets. In contrast, 5D2 and CR1 completely inhibited asialo-vWf–induced platelet aggregation and ristocetin-induced vWf binding to GP Ib-IX-V. However, only 5D2 blocked botrocetin- and jaracetin-induced vWf binding to platelets and binding of vWf to botrocetin- and jaracetin-coated beads. Epitopes for 5D2 and CR1 were conformationally dependent, but not congruent. Other antibodies mapped to epitopes within the A1 domain (CR2 and CR15, Leu-494–Leu-512; CR2, Phe-536–Ala-554; CR3, Arg-578–Glu-596; CR11 and CR15, Ala-564–Ser-582) were not functional, identifying regions of the vWf A1 domain not directly involved in vWf-GP Ib-IX-V interaction. The combined results provide evidence that the proline-rich sequence Glu-700–Asp-709 constitutes a regulatory site for ristocetin, and that ristocetin and botrocetin induce, at least in part, separate receptor-recognition sites on vWf. (Blood. 2000;95:164-172)

scholarly journals Asialo-von Willebrand factor inhibits platelet adherence to human arterial subendothelium: discrepancy between ristocetin cofactor activity and primary hemostatic function

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1084-1089 ◽  
Author(s):  
JB Lawrence ◽  
HR Gralnick
Keyword(s):  
Von Willebrand Factor ◽  
Whole Blood ◽  
Specific Activity ◽  
Human Platelets ◽  
Primary Hemostasis ◽  
Platelet Adherence ◽  
Wall Shear ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

Abstract Platelet adherence at high wall shear rates requires plasma von Willebrand factor (vWF). Clinically, the ristocetin cofactor (RCof) activity is the only widely available assay for vWF function. When purified vWF is treated with neuraminidase to yield asialo-vWF (AS- vWF), its RCof activity is increased by 20% to 40%. AS-vWF binds to normal human platelets independently of ristocetin and induces platelet aggregation in the presence of fibrinogen. To determine whether AS-vWF also shows an enhanced capacity to support platelet adherence to subendothelium, we used the Baumgartner technique. Intact vWF, AS-vWF, or AS-vWF treated with beta-galactosidase (asialo, agalacto-vWF; AS,AG- vWF) was added to normal citrated whole blood before perfusion over human umbilical artery segments (wall shear rate, 2,600 sec-1). Four micrograms per milliliter AS-vWF caused a 69% reduction in total platelet adherence compared with citrated whole blood (P less than .001), and 4 micrograms/mL AS,AG-vWF led to a 48% reduction (P less than .005). With 4 micrograms/mL intact vWF, the platelet adherence values were not significantly different from the controls. No significant differences in subendothelial platelet thrombi or postperfusion platelet counts were evident among any of the groups. In reconstituted afibrinogenemic perfusates, 4 micrograms/mL AS-vWF caused a 42% reduction in platelet adherence (P less than .05). Thus, AS-vWF is a potent inhibitor of platelet adherence, despite its enhanced RCof specific activity. Abnormalities in vWF carbohydrate may play a role in impaired primary hemostasis in some patients with von Willebrand's disease.

scholarly journals A new congenital platelet abnormality characterized by spontaneous platelet aggregation, enhanced von Willebrand factor platelet interaction, and the presence of all von Willebrand factor multimers in plasma

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2028-2033
Author(s):  
A Casonato ◽  
L De Marco ◽  
M Mazzucato ◽  
V De Angelis ◽  
D De Roia ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Von Willebrand Disease ◽  
Bleeding Tendency ◽  
Binding Studies ◽  
Spontaneous Platelet Aggregation ◽  
Von Willebrand ◽  
Willebrand Factor

A case is reported of a 49-year-old woman with a mild bleeding tendency. Her bleeding time, platelet count and size, plasma ristocetin cofactor activity, von Willebrand factor (vWF) antigen, and vWF multimeric pattern are all within normal limits. Spontaneous platelet aggregation is observed when citrated platelet-rich plasma (PRP) is stirred in an aggregometer cuvette. This aggregation is completely is only slightly diminished by an antiglycoprotein (GP) IIb/IIIa or by an anti GPIb monoclonal antibody. The patient's PRP shows increased sensitivity to ristocetin. The distinct feature of this patient, also present in two family members studied, is that platelet aggregation is initiated by purified vWF in the absence of any other agonist. The vWF- induced platelet aggregation is abolished by anti-GPIb and anti- GPIIb/IIIa monoclonal antibodies and by EDTA (5 mmol/L). Apyrase inhibits the second wave of aggregation. Patient's platelets in PRP are four to six times more reactive to asialo vWF-induced platelet aggregation than normal platelets. The amount of radiolabeled vWF bound to platelets in the presence of either low concentration of ristocetin or asialo vWF was increased 30% compared with normal. The patient's platelet GPIb was analyzed by SDS page and immunoblotting and by binding studies with anti-GPIb monoclonal antibodies showed one band with slightly increased migration pattern and a normal number of GPIb molecules. Unlike the previously reported patients with pseudo or platelet-type von Willebrand disease, this patient has normal vWF parameters.

scholarly journals Factor VIII-von Willebrand factor requires calcium for facilitation of platelet adherence

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 996-103 ◽  
Author(s):  
KS Sakariassen ◽  
M Ottenhof-Rovers ◽  
JJ Sixma
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Divalent Cations ◽  
Platelet Adherence ◽  
Human Arteries ◽  
Platelet Spreading ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

The role of divalent cations in platelet adherence to deendothelialized human arteries in flowing blood was investigated in an annular perfusion chamber. Spreading of platelets on the subendothelium was impaired below 30 microM of free Ca2+ ions (Ca2+). When Ca2+ was replaced by Mg2+, adherence was unchanged in perfusates without exogenous factor VIII-von Willebrand factor (FVIII-vWF), but the ability of FVIII-vWF to support platelet adherence was lost. Binding of FVIII-vWF to the vessel wall was independent of divalent cations, but bound FVIII-vWF was only able to mediate adherence after exposure to Ca2+. Pretreatment of FVIII-vWF with the calcium chelator EGTA (10 mM) resulted in loss of the ability to facilitate platelet adherence, while the ristocetin cofactor activity remained intact. Full restoration of the ability to mediate platelet adherence could only be obtained by prolonged dialysis against Ca2+ in the millimolar range. These data indicate that divalent cations have at least two separate roles to play in supporting platelet adherence: (1) platelet spreading on the subendothelium requires Ca2+ or Mg2+; (2) FVIII-vWF should be exposed to Ca2+ to obtain its optimal biologic activity in supporting platelet adherence.

Von Willebrand Factor Antigen and Factor XI Activity Levels As Predictors of Bleeding Tendency in Israeli Patients with Von Willebrand's Disease

1995 ◽  
Vol 1 (4) ◽  
pp. 260-264 ◽  
Author(s):  
Benjamin Brenner ◽  
Tamar Stemberg ◽  
Arieh Laor ◽  
Shulamit Tavori ◽  
Ilana Tatarsky ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Activity Levels ◽  
Factor Xi ◽  
Factor Xi Deficiency ◽  
Von Willebrand Factor Antigen ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor ◽  
Factor Antigen

Previous preliminary data and case reports have suggested an association of von Willebrand's disease (vWD) with factor XI deficiency and platelet abnormalities. We have analyzed the prevalence of factor XI deficiency and thrombocytopathy in a cohort of Israeli patients with vWD. Decreased factor XI levels (<67 U/dl) were documented in 35 of 63 (36%) vWD subjects; factor XI levels were <30 U/dl in five of 60 (8%). A significant decline in ADP-induced platelet aggregation (<30% of control) was found in 48% of vWD patients. Likewise, epinephrine-induced aggregation was reduced in 41%, and collagen-induced aggregation was decreased in 7% of vWD patients. Logistic regression analysis showed that while Ivy bleeding time, ristocetin cofactor, and ristocetin-induced platelet aggregation did not predict bleeding, both von Willebrand factor antigen and factor XI activity levels predict bleeding in patients with vWD. These findings suggest that mild factor XI deficiency and thrombocytopathy are common in Israeli subjects with vWD and that associated factor XI deficiency can result in clinical bleeding in these patients. Key Words: Von Willebrand' s disease—Factor XI deficiency—Thrombocytopathy.

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