scholarly journals A new von Willebrand variant (type I, New York): increased ristocetin- induced platelet aggregation and plasma von Willebrand factor containing the full range of multimers

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 149-156 ◽  
Author(s):  
HJ Weiss ◽  
II Sussman
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Full Range ◽  
Sodium Dodecyl ◽  
Initial Slope ◽  
Second Phase ◽  
Type I ◽  
Von Willebrand ◽  
Willebrand Factor

We report three members of a family who had reduced levels of plasma von Willebrand factor (vWF) and increased ristocetin-induced platelet aggregation (RIPA) (aggregation of platelet-rich plasma with ristocetin at a concentration of 0.45 mg/mL), as previously reported in type IIB and pseudo-von Willebrand's disease (vWD). However, in contrast to the latter two disorders in which the larger vWF multimers are absent in plasma, the entire range of vWF multimers was observed in the patients' plasma after sodium dodecyl sulfate-agarose gel electrophoresis, and all vWF multimers (including the largest) were present in the same proportion as in normal plasma and type I vWD. Thus, despite increased RIPA, the levels and multimeric pattern of vWF in this family's plasma were indistinguishable from those in type I vWD in which RIPA is usually decreased. Addition of ristocetin to the patients' platelet- rich plasma resulted in the removal of vWF (and, more selectively, of the large multimers) at lower concentrations of ristocetin than normal, as in type IIB and pseudo-vWD. The defect in the patients was localized to their vWF, which had an enhanced capacity for aggregating washed normal platelets in the presence of low concentrations of ristocetin and for aggregating pseudo-vWD platelets (in the absence of ristocetin). Both glycoproteins (GP) Ib and IIb-IIIa were involved in the enhanced aggregation response. RIPA (at low ristocetin concentrations) in the patients' platelet-rich plasma was abolished by a monoclonal antibody (AP1) to GPIb and was markedly reduced by monoclonal antibodies (10E5 and LJP9) that block adenosine diphosphate and thrombin-induced binding of vWF and fibrinogen to GPIIb-IIIa but was unaffected by an antibody (LJP5) that only blocks vWF binding. Partial inhibition of the initial aggregation slope (and complete inhibition of second phase aggregation) was achieved with creatine phosphate/creatine phosphokinase. EDTA blocked second-phase aggregation but was without effect on the initial slope. The findings in this family combine some features of both type I vWD (normal pattern of vWF multimers in plasma) and type IIB vWD (increased RIPA) and further demonstrate the increasing complexity of the structure-function relationships in vWD.

scholarly journals A new von Willebrand variant (type I, New York): increased ristocetin- induced platelet aggregation and plasma von Willebrand factor containing the full range of multimers

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 149-156 ◽  
Author(s):  
HJ Weiss ◽  
II Sussman
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Full Range ◽  
Sodium Dodecyl ◽  
Initial Slope ◽  
Second Phase ◽  
Type I ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We report three members of a family who had reduced levels of plasma von Willebrand factor (vWF) and increased ristocetin-induced platelet aggregation (RIPA) (aggregation of platelet-rich plasma with ristocetin at a concentration of 0.45 mg/mL), as previously reported in type IIB and pseudo-von Willebrand's disease (vWD). However, in contrast to the latter two disorders in which the larger vWF multimers are absent in plasma, the entire range of vWF multimers was observed in the patients' plasma after sodium dodecyl sulfate-agarose gel electrophoresis, and all vWF multimers (including the largest) were present in the same proportion as in normal plasma and type I vWD. Thus, despite increased RIPA, the levels and multimeric pattern of vWF in this family's plasma were indistinguishable from those in type I vWD in which RIPA is usually decreased. Addition of ristocetin to the patients' platelet- rich plasma resulted in the removal of vWF (and, more selectively, of the large multimers) at lower concentrations of ristocetin than normal, as in type IIB and pseudo-vWD. The defect in the patients was localized to their vWF, which had an enhanced capacity for aggregating washed normal platelets in the presence of low concentrations of ristocetin and for aggregating pseudo-vWD platelets (in the absence of ristocetin). Both glycoproteins (GP) Ib and IIb-IIIa were involved in the enhanced aggregation response. RIPA (at low ristocetin concentrations) in the patients' platelet-rich plasma was abolished by a monoclonal antibody (AP1) to GPIb and was markedly reduced by monoclonal antibodies (10E5 and LJP9) that block adenosine diphosphate and thrombin-induced binding of vWF and fibrinogen to GPIIb-IIIa but was unaffected by an antibody (LJP5) that only blocks vWF binding. Partial inhibition of the initial aggregation slope (and complete inhibition of second phase aggregation) was achieved with creatine phosphate/creatine phosphokinase. EDTA blocked second-phase aggregation but was without effect on the initial slope. The findings in this family combine some features of both type I vWD (normal pattern of vWF multimers in plasma) and type IIB vWD (increased RIPA) and further demonstrate the increasing complexity of the structure-function relationships in vWD.

scholarly journals Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 927-937
Author(s):  
FM LaDuca ◽  
RE Bettigole ◽  
WR Bell ◽  
EB Robson
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Platelet Membrane ◽  
Type I ◽  
Membrane Glycoproteins ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.

scholarly journals Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 927-937 ◽  
Author(s):  
FM LaDuca ◽  
RE Bettigole ◽  
WR Bell ◽  
EB Robson
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Platelet Membrane ◽  
Type I ◽  
Membrane Glycoproteins ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.

scholarly journals New Method for Assessment of Plasma Von Willebrand Factor

1977 ◽  
Author(s):  
T. Sano ◽  
T. Motomiya ◽  
N. Mashimo ◽  
H. Yamazaki
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Normal Subjects ◽  
Induce Platelet Aggregation ◽  
Platelet Suspension ◽  
Maximal Plasma ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Diabetes Melitus

As much interests have been focused on von Willebrand factor (vWF) in diabetes melitus and atherosclerosis, request to determine vWF has been increasing recently. Two methods for assessment of plasma vWF level, without platelet aggregometer, were devised. 1) Platelet-rich plasma (PRP) sensitivity to ristocetin-induced platelet aggregation (RIPA): PRP was separated without centrifugation from citrated blood. Serially two-fold diluted restocetin (16 to 16x2-10 mg/ml) was prepared in a Cooke Microtiter tray and PRP (25 μl each) was added to each concentration of ristocetin. Then the ristocetin-PRP mixture was agitated for 15 seconds using a Kowa Kizai Micromixer and the minimum effective final concentration of ristocetin to give platelet aggregation was obtained microscopically and this was defined as PRP sensitivity to RIPA. This method is convenient for screening test. 2) vWF assay:Serially two-fold diluted plasma (2 to 1024 times, in Tris-salin pH 7.2 containing 12 mg/ml bovine serum albumin), fixed and washed platelet suspension (6x105 /μl, Macfarlane et al.1975) and 3 mg/ml ristocetin were mixed (25 μl each) in a microtiter tray and agitated for 15 seconds. The maximal plasma dilution to induce platelet aggregation was obtained microscopically and defined as the titer of plasma vWF. In normal subjects, minimum effective ristocetin concentration (PRP sensitivity to RIPA) was around 1 to 0.5 mg/ml and maximal plasma dilution to give platelet aggregation (vWF titer) was around 16 to 32 times. The present methods have a good reproducibility and are performed easily without aggregometer and thought to be useful clinically.

scholarly journals Further characterization of platelet-type von Willebrand's disease in Japan

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1254-1262 ◽  
Author(s):  
H Takahashi ◽  
M Handa ◽  
K Watanabe ◽  
Y Ando ◽  
R Nagayama ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Sodium Dodecyl ◽  
Membrane Glycoproteins ◽  
Glycoprotein Ib ◽  
Normal Platelet ◽  
Platelet Receptor ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We studied four patients who showed aggregation of platelets in platelet-rich plasma at lower concentrations of ristocetin than those required for normal platelet-rich plasma and who demonstrated an increased capacity of the platelets to bind normal von Willebrand factor. The four patients were from two Japanese families. Platelets from one family aggregated spontaneously in vitro, and platelets from both families aggregated upon the addition of normal plasma and cryoprecipitate, in the absence of ristocetin or other agonists. Analysis of the multimeric composition of von Willebrand factor by sodium dodecyl sulfate-agarose gel electrophoresis revealed a decrease in large multimers or a decrease in both large and intermediate multimers in plasma, but normal multimers in platelets. 1-Deamino-[8-D- arginine]-vasopressin caused by an immediate appearance of larger multimers in plasma, followed by the rapid disappearance of these multimers from circulating plasma. Analysis of platelet membrane glycoproteins from the patients showed that there were two distinct bands in the glycoprotein I region; one migrated in a slower region and the other in a faster region than normal glycoprotein Ib. We suggest that the platelet receptor abnormality in these patients is related to this abnormality of glycoprotein Ib.

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 Nanobody Activators of Von Willebrand Factor Via Targeting Its Autoinhibitory Module

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2074-2074
Author(s):  
Nicholas A Arce ◽  
Ally J Su ◽  
Renhao Li
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Binding Affinities ◽  
Yeast Display ◽  
E Coli ◽  
Platelet Receptor ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Introduction: Von Willebrand factor (VWF) is a multimeric plasma glycoprotein responsible for platelet arrest during injury, especially at high shear. After immobilization to the vessel wall, a VWF multimer is unfurled and elongated. This leads to exposure of the A1 domain therein that in turn binds to platelet receptor GPIbα and starts the aggregation process. Recently, it was suggested that VWF activation involves force-dependent disruption of the autoinhibitory module (AIM) that flanks the A1 domain on both sides. In this scenario, the AIM could be targeted for both VWF inhibition (Caplacizumab) and activation (ristocetin), although the exact mechanism and binding site of ristocetin still remains murky. If the quasi-stable structure of the AIM is important to VWF autoinhibition, specific disruption of its confirmation may be able to activate VWF. To this end, we sought to identify AIM-targeting activators using yeast surface display of a llama nanobody library. Methods: One adult Lama glama was immunized with recombinant human VWF AIM-A1 protein produced from transfected Expi293F cells. VHH specific genes were amplified from cDNAs prepared from PBMCs of the animal and electroporated into EBY100 cells. The resulting yeast display library was screened for AIM-specific binders via selection against binding to recombinant A1 protein without an intact AIM, and then for binding to the complex of AIM-A1 with GPIbα. Positive hits were produced as His-tagged monomeric nanobodies in E. coli and purified with nickel-affinity and gel filtration chromatography. The affinity of nanobodies to AIM-A1 was determined using bio-layer interferometry. Platelet-rich plasma from healthy donors was used to assess the effect of nanobodies on platelet aggregation in a light transmission aggregometer with comparison to that of ristocetin. Results: An AIM-A1-specific nanobody yeast display library was established. Several rounds of flow cytometry-based cell sorting of yeast cells with aforementioned binding properties produced AIM-binding nanobodies. Nanobodies encoded in three single clones have been expressed from E. coli and they exhibited differential binding affinities towards AIM-A1. Clone 6C4 showed the lowest affinity (K D 120 ± 3 nM), 6D12 showed intermediate affinity (K D 31 ± 0.8 nM), and 6C11 showed the highest affinity (K D 13.5 ± 0.2 nM) as shown in Figure 1. These nanobodies showed no detectable affinity towards recombinant A1-CAIM protein (residues 1268-1493), indicating that their epitopes are located in the N-terminal portion of the AIM (residues 1238-1267). When added to human platelet-rich plasma, each nanobody dose-dependently activated platelets and rapidly induced full platelet aggregation at concentrations exceeding the affinity of the nanobody for VWF (Figure 2). The aggregation could be inhibited by the addition of antibodies that block the interaction between VWF and GPIbα. Plots of extents of aggregation as a function of nanobody concentration produced EC 50 values of ~100 nM for 6C11 and 6D12. Conclusion: By isolating nanobodies that can bind specifically to the AIM and activate plasma VWF, we add supporting evidence that the AIM protects the A1 domain from binding to platelets. Interestingly, these nanobodies bind to the NAIM, on the opposite side of the module compared to ristocetin, the only known AIM-activating agent until now. With higher VWF-binding affinities than ristocetin and a robust profile as stable monomers, these nanobodies may prove useful in VWF-related research and diagnostics. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals The complex multimeric composition of factor VIII/von Willebrand factor

Blood ◽  
1981 ◽  
Vol 57 (6) ◽  
pp. 1140-1143 ◽  
Author(s):  
ZM Ruggeri ◽  
TS Zimmerman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Sodium Dodecyl ◽  
Factor Vii ◽  
Buffer System ◽  
Type I ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Von Willebrand ◽  
Willebrand Factor

We have analyzed the multimeric structure of factor VIII/von Willebrand factor in plasma by sodium dodecyl sulfate electrophoresis using gels of varying porosity and a discontinuous buffer system. Factor VIII/von Willebrand factor bands were identified by reaction with 125I-labeled affinity-purified antibody and subsequent autoradiography. In 1% agarose gels, normal plasma displayed a series of sharply defined oligomers. However, increasing the agarose concentration to 2.0% or utilizing mixtures of 0.8% agarose--1.75% acrylamide revealed two bands of lesser intensity interposed between the major bands. When the acrylamide concentration in the gels was increased to 2.5%, bands with a faster mobility than IgM and fibronectin were now evident. Type IIA von Willebrand's disease showed not only an absence of the larger multimers but also a relative increase in several of the newly identified bands as compared to type IIB, type I, and normal. These studies suggest that factor VII/von Willebrand factor in IIA von Willebrand's disease is structurally different from that in other forms of the disorder. They also indicate that the multimeric composition of factor VII/von Willebrand factor is more complex than can be explained by simple linear polymerization of a single protomer.

Purification of human von Willebrand factor–cleaving protease and its identification as a new member of the metalloproteinase family

Blood ◽  
2001 ◽  
Vol 98 (6) ◽  
pp. 1662-1666 ◽  
Author(s):  
Kazuo Fujikawa ◽  
Hiroshi Suzuki ◽  
Brad McMullen ◽  
Dominic Chung
Keyword(s):  
Amino Acid ◽  
Von Willebrand Factor ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Single Letter ◽  
Type I ◽  
Commercial Preparation ◽  
Amino Terminal ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (vWF) is synthesized in megakaryocytes and endothelial cells as a very large multimer, but circulates in plasma as a group of multimers ranging from 500 to 10 000 kd. An important mechanism for depolymerization of the large multimers is the limited proteolysis by a vWF-cleaving protease present in plasma. The absence or inactivation of the vWF-cleaving protease results in the accumulation of large multimers, which may cause thrombotic thrombocytopenic purpura. The vWF-cleaving protease was first described as a Ca++-dependent proteinase with an apparent molecular weight of approximately 300 kd. Thus far, however, it has not been isolated and characterized. In this study, the purification of human vWF-cleaving protease from a commercial preparation of factor VIII/vWF concentrate by means of several column chromatographic steps, including 2 steps of heparin-Sepharose column, is reported. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the anion exchange and gel filtration column fractions showed that the vWF-cleaving protease activity corresponded to a protein band of 150 kd. After reduction, it migrated with an apparent weight of 190 kd. The amino terminal sequence of the 150-kd band was AAGGIL(H)LE(L)L(D)AXG(P)X(V)XQ (single-letter amino acid codes), with the tentative residues shown in parentheses. A search of the human genome sequence identified the vWF-cleaving protease as a new member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motif) family of metalloproteinase. An active site sequence of HEIGHSFGLEHE (single-letter amino acid codes) was located at 150 residues from the N terminus of the protein.

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