scholarly journals Willebrand factor in hemostasis in the in vitro bleeding time

Blood ◽  
1979 ◽  
Vol 54 (6) ◽  
pp. 1347-1357 ◽  
Author(s):  
A Kimura ◽  
EJ Bowie ◽  
RJ Campbell ◽  
DN Fass
Keyword(s):  
Blood Volume ◽  
Bleeding Time ◽  
Normal Blood ◽  
In Vitro System ◽  
Skin Changes ◽  
Incision Site ◽  
Flow Through ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Heparinized porcine blood and plasma, at constant hydrostatic pressure, was allowed to flow through a 5-mm incision in a small piece of porcine skin. Changes in the exuded blood volume were measured, and the incision site was examined microscopically. When normal blood flowed through either normal or von Willebrand skin, the exuded blood volume decreased gradually and eventually stopped. Microscopic examination revealed a platelet plug in the incision site. This plug was positive for Willebrand factor when examined by immunofluorescence. In contrast, the blood from von Willebrand pigs continued to flow constantly, and a platelet plug was not seen. The delayed in vitro hemostasis in von Willebrand blood was corrected to the normal range by the addition of either normal plasma or partially purified Willebrand factor. Normal blood, in which the Willebrand factor was immunologically inhibited, showed delayed hemostasis. For this in vitro system, it appeared that plasmatic Willebrand factor played an essential role in hemostasis.

scholarly journals Willebrand factor in hemostasis in the in vitro bleeding time

Blood ◽  
1979 ◽  
Vol 54 (6) ◽  
pp. 1347-1357
Author(s):  
A Kimura ◽  
EJ Bowie ◽  
RJ Campbell ◽  
DN Fass
Keyword(s):  
Blood Volume ◽  
Bleeding Time ◽  
Normal Blood ◽  
In Vitro System ◽  
Skin Changes ◽  
Incision Site ◽  
Flow Through ◽  
Von Willebrand ◽  
Willebrand Factor

Heparinized porcine blood and plasma, at constant hydrostatic pressure, was allowed to flow through a 5-mm incision in a small piece of porcine skin. Changes in the exuded blood volume were measured, and the incision site was examined microscopically. When normal blood flowed through either normal or von Willebrand skin, the exuded blood volume decreased gradually and eventually stopped. Microscopic examination revealed a platelet plug in the incision site. This plug was positive for Willebrand factor when examined by immunofluorescence. In contrast, the blood from von Willebrand pigs continued to flow constantly, and a platelet plug was not seen. The delayed in vitro hemostasis in von Willebrand blood was corrected to the normal range by the addition of either normal plasma or partially purified Willebrand factor. Normal blood, in which the Willebrand factor was immunologically inhibited, showed delayed hemostasis. For this in vitro system, it appeared that plasmatic Willebrand factor played an essential role in hemostasis.

Antithrombotic Effect of a Recombinant von Willebrand Factor, VCL, on Nitrogen Laser-Induced Thrombus Formation in Guinea Pig Mesenteric Arteries

1995 ◽  
Vol 73 (02) ◽  
pp. 318-323 ◽  
Author(s):  
K Azzam ◽  
L I Garfinkel ◽  
C Bal dit Sollier ◽  
M Cisse Thiam ◽  
L Drouet
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Mesenteric Arteries ◽  
Antithrombotic Effects ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryTo assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded.In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i. v. injection inhibits ristocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time. In vivo, VCL (0.5 to 2 mg/kg) had no effect on the appearance of the thrombi formed but produced dose-dependent inhibition of the mean number of recurrent thrombi (the maximal effect was obtained at 5 min following i. v. injection of the highest dose: 0.8 ± 0.2 thrombi versus 4 ± 0.4 thrombi in controls). The three doses of VCL increased the time in which the first thrombus in a concentration-dependent manner was formed. However, the time to visualize the first thrombus was only prolonged in the higher dose-treated group.These in-vivo studies confirm that VCL induces immediate, potent, and transient antithrombotic effects. Most importantly, this inhibition was achieved without inducing thrombocytopenia nor prolongation of the bleeding time.

The Effect Of Antiporcine Willebrand Factor Monoclonal Antibodies On The Bleeding Time

1981 ◽  
Author(s):  
E J W Bowie ◽  
D N Fass ◽  
J A Katzmann
Keyword(s):  
Monoclonal Antibodies ◽  
Bleeding Time ◽  
Vitro Assay ◽  
Functional Sites ◽  
Antibody Titers ◽  
Vitro Model ◽  
Von Willebrand ◽  
Willebrand Factor

Murine monoclonal antibodies to porcine Willebrand factor were used to study the role of Willebrand factor in hemostatic plug formation in the in vivo and in vitro skin bleeding times. The in vivo assay requires the intravenous injection of antibody-containing mouse ascitic fluids into normal animals with subsequent ear bleeding time tests performed over a 24-hour period. The in vitro assay was carried out with heparinized normal porcine blood flowing through a 0.5 cm incision in an excised piece of porcine skin. The heat inactivated ascitic fluids, containing the antibodies, were used ;Ln these assays at final dilutions of 2 × 102 through 2 × 105 The 7 antibodies used comprised at least 5 groups with differing reactivities based on assays other than bleeding time tests. The antibody titers in a poreineg Willebrand factor-binding radioimmunoassay ranged from 108 through 1012 . Doses of 10HL of ascitic fluid/kg of body weight (approximately 2 × 103 final dilution) were able to transiently prolong the in vivo bleeding time without alteration of other VIII complex values (VIII:C, ristocetin cofactor activity and Vlll-related antigen). Higher doses resulted in bleeding times similar to von Willebrand pigs (>15 min) immediately following infusion, with a decay of the effect over the next 2 hours. At dilutions of 2 × 104 selected monoclonal antibodies interfered with hemostasis in the in vitro model whereas other antibodies inhibited only at 2 × 102 dilution. With one exception, the potencies of the antibodies appeared to be similar in both assays. The titer of the antibodies in the radioimmunoassay does not appear to correlate with prolongation of the in vitro bleeding time, suggesting specific reactivity with functional sites involved in hemostatic plug formatio.

scholarly journals Subunit composition of plasma von Willebrand factor in patients with the myeloproliferative syndrome

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1213-1217 ◽  
Author(s):  
U Budde ◽  
JA Dent ◽  
SD Berkowitz ◽  
ZM Ruggeri ◽  
TS Zimmerman
Keyword(s):  
Von Willebrand Factor ◽  
Bleeding Time ◽  
Polyacrylamide Gel Electrophoresis ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Prolonged Bleeding Time ◽  
Myeloproliferative Syndrome ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In order to evaluate the role of proteolysis in acquired von Willebrand's disease (vWD) associated with the myeloproliferative syndrome, we have determined the relative quantity of von Willebrand factor (vWF) fragments as compared with the intact 225 kDa subunit in four patients. The plasma vWF of each individual lacked large multimers; each had a prolonged bleeding time; and both platelet and leukocyte counts were elevated. Plasma was obtained from blood drawn into 1 mmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 5 mmol/L EDTA to prevent in vitro proteolysis. vWF was isolated from plasma by immunoadsorbent chromatography, reduced, subjected to SDS-5% polyacrylamide gel electrophoresis, and immunoblotted with a mixture of 55 anti-vWF monoclonal antibodies. In three patients with essential thrombocytosis (ET) the 176 and 140 kDa fragments were increased in proportion to the intact 225 kDa subunit indicating increased proteolysis. Treatment of one ET patient with CCNU (Lomustine) decreased the platelet count and, to a lesser extent, the white blood cell count. This was associated with a correction of the bleeding time, a partial correction of the multimeric abnormality, and a lessening of vWF cleavage. In a patient with polycythemia rubra vera (PRV) the proportion of the 176 kDa fragment was increased to the upper limit of normal but there was no definite evidence of increased proteolysis. These studies provide evidence that proteolysis plays a role in the acquired von Willebrand's disease associated with the myeloproliferative syndrome. However, other mechanisms must also be considered.

Association Between Bleeding Time and Platelet Adherence to Artery Subendothelium

1984 ◽  
Vol 52 (02) ◽  
pp. 144-147 ◽  
Author(s):  
K S Sakariassen ◽  
P A Bolhuis ◽  
Margaretha Blombäck ◽  
L Thorell ◽  
Birger Blombäck ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Bleeding Time ◽  
Blood Platelets ◽  
Normal Subjects ◽  
Commercial Preparation ◽  
Normal Platelet ◽  
Platelet Adherence ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe efficacy of five different factor VUI-von Willebrand factor (FVIII-VWF) preparations in mediating adherence of blood platelets to damaged vessel walls was tested in an annular perfusion chamber utilizing human arteries and reconstituted blood.FVIII-VWF-purified by Sepharose CL-4B chromatography and von Willebrand factor prepared from this preparation by dissociation with 0.25 M CaCl2 followed by Sepharose CL-6B chromatography were equally effective in mediating platelet adherence as FVIII-VWF in cryoprecipitate and in plasma from normal subjects. A commercial concentrate of FVIII-VWF (Hemofil, Hyland) used for the treatment of haemophiliacs did not mediate platelet adherence at normal levels of FVIII-VWF related properties. A recently developed high-purity FVIII-VWF preparation (Concentrate II) containing multimers of high molecular weight normalized the platelet adherence. Platelet adherence in plasma obtained from two patients with von Willebrand’s disease (VWD) was impaired, but plasma samples obtained following treatment with Concentrate II mediated normal platelet adherence. The normalization of platelet adherence paralleled the normalization of the bleeding time.This platelet adherence assay offers an inexpensive and efficient in vitro tool to test the efficacy of FVIII-VWF preparations designed for VWD patients. Preparations such as cryoprecipitate and Concentrate II mediated the platelet adherence and normalized the bleeding time. The commercial preparation did not mediate platelet adherence and had no effect on the bleeding time.

scholarly journals The heterogeneity of type IIA von Willebrand's disease: studies with protease inhibitors

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1207-1212 ◽  
Author(s):  
J Batlle ◽  
MF Lopez Fernandez ◽  
M Campos ◽  
B Justica ◽  
C Berges ◽  
...  
Keyword(s):  
Bleeding Time ◽  
Relative Proportion ◽  
Clinical Expression ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Time Correction ◽  
Von Willebrand ◽  
Willebrand Factor

The absence of large von Willebrand factor (vWF) multimers from plasma is a characteristic of Type IIA von Willebrand's disease (vWD) and is thought to contribute to the clinical expression of this disorder. Recently, three IIA patients have been reported in whom intermediate and large multimers could be restored if blood were collected in 5 mm EDTA, 6 mmol/L N-ethylmaleimide, and 1 mmol/L leupeptin. This suggested that absence of large multimers resulted from in vitro proteolysis. We have now collected blood from ten Type IIA vWD patients in these inhibitors but were not able to detect large multimers in the plasma of any of them. In addition, intermediate-sized multimers were reduced or completely absent in all. The inclusion of inhibitors in the citrate anticoagulant, as compared to citrate alone, was found to increase the relative proportion of intermediate multimers in some patients but had no effect in others, and in none did it restore large multimers to plasma. The results with platelet vWF were more varied. Four patients showed an absence or decrease of large multimers, whereas in seven patients large multimers were present. When compared with citrate anticoagulant alone, the inclusion of inhibitors in the anticoagulant had little or no effect on the platelet multimeric pattern. 1-Deamino-8- D-Arginine Vasopressin (DDAVP) was administered to six patients from five families. Two patients from one family showed complete correction and a third patient showed almost complete correction of her bleeding time. Two patients showed minimal correction and one showed no detectable correction. An increase in multimer size after DDAVP tended to be associated with correction of the bleeding time. However, in no case did the largest multimers appear in plasma even in patients with complete bleeding time correction. The presence or absence of inhibitors in the anticoagulant had little or no effect on the multimeric pattern after DDAVP. These results indicate that Type IIA vWD is a heterogeneous disorder in which absence of largest and intermediate multimers is an in vivo phenomenon.

scholarly journals Subunit composition of plasma von Willebrand factor in patients with the myeloproliferative syndrome

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1213-1217 ◽  
Author(s):  
U Budde ◽  
JA Dent ◽  
SD Berkowitz ◽  
ZM Ruggeri ◽  
TS Zimmerman
Keyword(s):  
Von Willebrand Factor ◽  
Bleeding Time ◽  
Polyacrylamide Gel Electrophoresis ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Prolonged Bleeding Time ◽  
Myeloproliferative Syndrome ◽  
Von Willebrand ◽  
Willebrand Factor

In order to evaluate the role of proteolysis in acquired von Willebrand's disease (vWD) associated with the myeloproliferative syndrome, we have determined the relative quantity of von Willebrand factor (vWF) fragments as compared with the intact 225 kDa subunit in four patients. The plasma vWF of each individual lacked large multimers; each had a prolonged bleeding time; and both platelet and leukocyte counts were elevated. Plasma was obtained from blood drawn into 1 mmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 5 mmol/L EDTA to prevent in vitro proteolysis. vWF was isolated from plasma by immunoadsorbent chromatography, reduced, subjected to SDS-5% polyacrylamide gel electrophoresis, and immunoblotted with a mixture of 55 anti-vWF monoclonal antibodies. In three patients with essential thrombocytosis (ET) the 176 and 140 kDa fragments were increased in proportion to the intact 225 kDa subunit indicating increased proteolysis. Treatment of one ET patient with CCNU (Lomustine) decreased the platelet count and, to a lesser extent, the white blood cell count. This was associated with a correction of the bleeding time, a partial correction of the multimeric abnormality, and a lessening of vWF cleavage. In a patient with polycythemia rubra vera (PRV) the proportion of the 176 kDa fragment was increased to the upper limit of normal but there was no definite evidence of increased proteolysis. These studies provide evidence that proteolysis plays a role in the acquired von Willebrand's disease associated with the myeloproliferative syndrome. However, other mechanisms must also be considered.

A Patient with Acquired von Willebrand’s Syndrome and Platelet Aggregating Activity in the IgC Fraction

1975 ◽  
Author(s):  
J. W. ten Cate ◽  
R. Wanders ◽  
M. Schneider-Trip
Keyword(s):  
Von Willebrand Factor ◽  
Column Chromatography ◽  
Platelet Adhesion ◽  
Bleeding Time ◽  
Hemophilia A ◽  
Factor Activity ◽  
Platelet Antibodies ◽  
Von Willebrand ◽  
Willebrand Factor

A new case is described of acquired von Willebrand’s syndrome: bleeding time 20’; platelet-adhesion 20%; F. VIII 9% (procoag. act.); F. VIII R. A. not demonstrable; von Willebrand factor activity (Weiss assay) not demonstrable; absent Ristocetin aggregation. No anti-F. VIII activity could be demonstrated in vitro. Cryoprecipitate infusion resulted in low recovery of f. VIII activity and no f. VIII-synthesis. The serum contained a platelet aggregating activity when tested on normal PHP or washed platelets of 30 random selected donors, and also in PRP of hemophilia A or severe von Willebrand patients. The aggregating activity was present in serum, heated serum (30’ 56° C), BaSO4 absorbed serum, in the IgG fraction isolated both by column chromatography and by ultracentrifugation. Absorption of serum with Vo fraction (sepharose 6B) of normal cryoprecipitate did not remove the aggregating activity. Aggregation was not inhibited by aspirin, adenosine or heparin and was inhibited by EDTA and Cocain and independant of complement. The aggregation was characterized by serotonin and nucleotide release in comparable amounts as thrombin. E. M. studies revealed degranulation and viscous metamorphosis.The platelet aggregating activity is probably due to platelet antibodies.

Patients with isolated prolonged in vitro bleeding time

2011 ◽  
Vol 31 (S 01) ◽  
pp. S64-S68
Author(s):  
M. Stein ◽  
K. Scholz ◽  
B. Llugaliu ◽  
G. Asmelash ◽  
W. Miesbach ◽  
...  
Keyword(s):  
Platelet Function ◽  
Von Willebrand Factor ◽  
Bleeding Time ◽  
Von Willebrand Disease ◽  
Treatment Concept ◽  
Main Symptom ◽  
Platelet Function Disorders ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryIn patients with isolated prolonged in vitro bleeding time there is no standardised treatment concept. With this study we characterized the extent of bleeding symptoms. Patients, methods All diagnoses known to cause prolonged in vitro bleeding time (PFA-100) (epinephrine-cartridge >160 s, ADP-cartridge > 120 s) have been excluded, such as platelet function disorders, effects of medications, nutrition or von Willebrand disease. 75 patients (77%, n = 58 women; 23%, n = 17 men, median age 46 (16–81) years were included. All bleeding symptoms have been stored in a databank with help of a comprehensive questionnaire. Results 78% (n = 54) of all patients reported of having had an operation, 69.8% (n = 37) of them described postoperative bleedings (p = 0.0373). 13.5% (n = 5) of the 54 could remember having been randomly treated by the administration of a transfusion and only 2.7% (n = 1) were treated by substitution of von Willebrand factor. 71% (n = 51) patients indicated haematoma (p = 0.8116). About 33.8% (n = 24) patients had gum bleeding and 40.8% (n = 29, p = 0.7808) patients reported bleeding after the dentist. 41.4% (n = 29) patients suffered under frequent epistaxis (p = 0.0212). There was no correlation between prolonged epinephrine bleeding time to VWF : Ag (rho = 0.16) nor to VWF : RCo (rho = 0.12) nor between prolonged epinephrine and ADP bleeding time (rho = 0.01) nor to ROTEM® analysis. Conclusion Patients with isolated prolonged PFA are mainly women and can be affected by all kinds of bleedings while haematoma is the main symptom. VWD might not be causal

scholarly journals The heterogeneity of type IIA von Willebrand's disease: studies with protease inhibitors

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1207-1212 ◽  
Author(s):  
J Batlle ◽  
MF Lopez Fernandez ◽  
M Campos ◽  
B Justica ◽  
C Berges ◽  
...  
Keyword(s):  
Bleeding Time ◽  
Relative Proportion ◽  
Clinical Expression ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Time Correction ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The absence of large von Willebrand factor (vWF) multimers from plasma is a characteristic of Type IIA von Willebrand's disease (vWD) and is thought to contribute to the clinical expression of this disorder. Recently, three IIA patients have been reported in whom intermediate and large multimers could be restored if blood were collected in 5 mm EDTA, 6 mmol/L N-ethylmaleimide, and 1 mmol/L leupeptin. This suggested that absence of large multimers resulted from in vitro proteolysis. We have now collected blood from ten Type IIA vWD patients in these inhibitors but were not able to detect large multimers in the plasma of any of them. In addition, intermediate-sized multimers were reduced or completely absent in all. The inclusion of inhibitors in the citrate anticoagulant, as compared to citrate alone, was found to increase the relative proportion of intermediate multimers in some patients but had no effect in others, and in none did it restore large multimers to plasma. The results with platelet vWF were more varied. Four patients showed an absence or decrease of large multimers, whereas in seven patients large multimers were present. When compared with citrate anticoagulant alone, the inclusion of inhibitors in the anticoagulant had little or no effect on the platelet multimeric pattern. 1-Deamino-8- D-Arginine Vasopressin (DDAVP) was administered to six patients from five families. Two patients from one family showed complete correction and a third patient showed almost complete correction of her bleeding time. Two patients showed minimal correction and one showed no detectable correction. An increase in multimer size after DDAVP tended to be associated with correction of the bleeding time. However, in no case did the largest multimers appear in plasma even in patients with complete bleeding time correction. The presence or absence of inhibitors in the anticoagulant had little or no effect on the multimeric pattern after DDAVP. These results indicate that Type IIA vWD is a heterogeneous disorder in which absence of largest and intermediate multimers is an in vivo phenomenon.

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