scholarly journals Dependence of plasmin-mediated degradation of platelet adhesive receptors on temperature and Ca2+

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1546-1557
Author(s):  
KJ Winters ◽  
PR Eisenberg ◽  
AS Jaffe ◽  
SA Santoro
Keyword(s):  
Platelet Activation ◽  
Direct Consequence ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Membrane Glycoproteins ◽  
Type Plasminogen Activator ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Marked Dependence

The effects of activation of plasminogen by streptokinase and tissue- type-plasminogen activator on platelet activation and the membrane glycoproteins (GPs) that mediate platelet adhesion and aggregation are not yet fully defined. To clarify effects on platelets during activation of plasminogen in vitro, we used monoclonal antibodies (MoAbs), flow cytometry, and platelets surface-labeled with 125I to characterize changes in receptors for fibrinogen (GPIIb-IIIa), von Willebrand factor (GPIb), and collagen (GPIa-IIa). Activation of plasminogen in plasma with pharmacologic concentrations of plasminogen activators did not degrade GPIIb-IIIa or GPIb, and caused only a modest decrease in GPIa. In washed platelets GPIIb-IIIa was extensively degraded by plasmin at 37 degrees C in the absence of exogenous Ca2+, conditions that destabilize the IIb-IIIa complex. Degradation of GPIb in washed platelets displayed a similar although less-marked dependence on temperature and the absence of Ca2+. The binding of activation- specific MoAbs did not increase during activation of plasminogen in plasma. We conclude that during pharmacologic fibrinolysis, reported inhibition of platelet function in plasma is not due to degradation of platelet-adhesive receptors. In addition, platelet activation observed during thrombolytic therapy does not appear to be a direct consequence of plasminogen activation.

scholarly journals Dependence of plasmin-mediated degradation of platelet adhesive receptors on temperature and Ca2+

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1546-1557 ◽  
Author(s):  
KJ Winters ◽  
PR Eisenberg ◽  
AS Jaffe ◽  
SA Santoro
Keyword(s):  
Platelet Activation ◽  
Direct Consequence ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Membrane Glycoproteins ◽  
Type Plasminogen Activator ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Marked Dependence

Abstract The effects of activation of plasminogen by streptokinase and tissue- type-plasminogen activator on platelet activation and the membrane glycoproteins (GPs) that mediate platelet adhesion and aggregation are not yet fully defined. To clarify effects on platelets during activation of plasminogen in vitro, we used monoclonal antibodies (MoAbs), flow cytometry, and platelets surface-labeled with 125I to characterize changes in receptors for fibrinogen (GPIIb-IIIa), von Willebrand factor (GPIb), and collagen (GPIa-IIa). Activation of plasminogen in plasma with pharmacologic concentrations of plasminogen activators did not degrade GPIIb-IIIa or GPIb, and caused only a modest decrease in GPIa. In washed platelets GPIIb-IIIa was extensively degraded by plasmin at 37 degrees C in the absence of exogenous Ca2+, conditions that destabilize the IIb-IIIa complex. Degradation of GPIb in washed platelets displayed a similar although less-marked dependence on temperature and the absence of Ca2+. The binding of activation- specific MoAbs did not increase during activation of plasminogen in plasma. We conclude that during pharmacologic fibrinolysis, reported inhibition of platelet function in plasma is not due to degradation of platelet-adhesive receptors. In addition, platelet activation observed during thrombolytic therapy does not appear to be a direct consequence of plasminogen activation.

Assay of Functional Plasminogen in Rat Plasma Applicable to Experimental Studies of Thrombolysis

2000 ◽  
Vol 84 (08) ◽  
pp. 299-306 ◽  
Author(s):  
Kristian Bangert ◽  
Sixtus Thorsen
Keyword(s):  
Plasminogen Activator ◽  
Experimental Studies ◽  
Fold Increase ◽  
Rat Plasma ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Type Plasminogen Activator ◽  
Plasmin Inhibitor

SummaryAn improved sensitive, specific, precise and accurate assay of plasminogen in rat plasma was developed. It is performed in 96-well microtiter plates and can be completed within one hour. The assay is based on activation of plasminogen by human urokinase-type plasminogen activator (uPA) and simultaneous measurement of generated plasmin with the specific plasmin substrate H-D-Val-Phe-Lys-4-nitroanilide (S-2390), using purified native rat plasminogen for calibration. The concentration of S-2390 in the final reaction mixture during the whole reaction period is much greater than the K m value (≈20 µM) for rat plasmin-cleavage of S-2390 ensuring that hydrolysis of substrate follows zero order kinetics and that the substrate produces a 20-35 fold decrease in rate of inhibition of plasmin by its target inhibitors in plasma. Analogous to the human system the target plasma inhibitors of rat plasmin are shown to be plasmin inhibitor and α-macroglobulins. Tranexamic acid (0.8 mM) is incorporated in the reaction mixture resulting in a 19-fold increase in the rate of plasminogen activation and presumably an about 50-fold decrease in the rate of inhibition of generated plasmin by plasmin inhibitor. The assay is suitable for accurate measurement of plasminogen in samples obtained from animals containing pharmacological concentrations of uPA or tissue-type plasminogen activator (tPA) in their plasma when in vitro plasminogen activation is blocked at pH 5 by collecting blood in acidic anticoagulant. Judged from in vitro experiments formation of catalytic active plasmin-α-macroglobulin complexes during massive activation of plasminogen in vivo does not interfere with the assay.

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 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.

The Simultaneous Acute Release of Tissue-Type Plasminogen Activator and von Willebrand Factor in the Perfused Rat Hindleg Region

1990 ◽  
Vol 63 (03) ◽  
pp. 454-458 ◽  
Author(s):  
N Tranquille ◽  
J J Emeis
Keyword(s):  
Plasminogen Activator ◽  
Von Willebrand Factor ◽  
Time Course ◽  
Platelet Activating Factor ◽  
Calcium Ionophore ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryIn perfused rat hindlegs, platelet-activating factor and bradyki-nin induced the acute release of both tissue-type plasminogen activator (t-PA) and von Willebrand Factor (vWF). The time course of release was similar for both proteins, and the amounts of t-PA and vWF released under various conditions were closely correlated. Release of both t-PA and vWF required extracellular calcium, and could be induced by the calcium ionophore A-23187. Protein synthesis was not required for release to occur.Phorbol myristate acetate also induced release of t-PA and vWF, though with a different time course; DDAVP was inactive.The results suggest that the release of t-PA, and that of vWF, are closely linked at the cellular level.

The Role of Cyclic Nucleotides in the Release of Tissue-Type Plasminogen Activator and von Willebrand Factor

1993 ◽  
Vol 69 (03) ◽  
pp. 259-261 ◽  
Author(s):  
N Tranquille ◽  
J J Emeis
Keyword(s):  
Plasminogen Activator ◽  
Von Willebrand Factor ◽  
Cyclic Nucleotides ◽  
Atrial Natriuretic Factor ◽  
Platelet Activating Factor ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe modulation of the induced acute release of tissue-type plasminogen activator (t-PA) and of von Willebrand factor (vWF) by compounds affecting cyclic nucleotide levels was studied, using an isolated rat hindleg perfusion system. Platelet-activating factor (PAF; 5 nM) or bradykinin (0.8 (μM) were used to induce release of t-PA and vWF.The guanylate cyclase activators sodium nitroprusside and atrial natriuretic factor reduced the induced release of t-PA and vWF. Release was not affected by inhibiting nitric oxide production with NG-nitro-L-arginine. The effects of nitroprusside and atrial natriuretic factor could not be reproduced by infusion of 8-bromo-cGMP.The adenylate cyclase activator forskolin had no effect on bradykinin-induced release of t-PA and vWF, reduced PAF-induced t-PA release, but potentiated PAF-induced vWF release. These modulatory effects were only partially mimicked by infusion of 8-bromo-cAMP.None of the compounds tested was able to induce the release of t-PA or of vWF in the absence of stimulation by bradykinin or platelet-activating factor. Cyclic nucleotides can thus modulate, but not induce, the acute release of t-PA and vWF from perfused rat hindlegs.

scholarly journals A monoclonal antibody preventing binding of tissue-type plasminogen activator to fibrin: useful to monitor fibrinogen breakdown during t-PA infusion

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1482-1487 ◽  
Author(s):  
P Holvoet ◽  
HR Lijnen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Plasminogen Activation ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Additional Support

Abstract One (MA-1C8) of 36 monoclonal antibodies obtained by fusion of P3X63- Ag8–6.5.3 myeloma cells with spleen cells of mice immunized with purified human tissue-type plasminogen activator (t-PA) blocked the activity of t-PA on fibrin plates but not on chromogenic substrates. MA- 1C8 at a concentration of 200 micrograms/mL inhibited plasma clot lysis and binding of t-PA to the clot. MA-1C8 had no influence on the activation of plasminogen by t-PA, which obeys Michaelis-Menten kinetics with Km = 105 mumol/L and kcat = 0.05 s-1; however, it abolished the influence of CNBr-digested fibrinogen on Km. These findings confirm that the stimulatory effect of fibrin on the activation of plasminogen by t-PA is mediated by binding of t-PA to fibrin and provide additional support for the kinetic model. Addition of t-PA to pooled fresh human plasma to a concentration of 5 micrograms/mL resulted in extensive fibrinogen breakdown after incubation for one hour at 37 degrees C or during storage at -20 degrees C for one day. In both instances, fibrinogen degradation was completely prevented by addition of MA-1C8 to a concentration of 200 micrograms/mL of plasma. MA-1C8 also effectively prevented in vitro fibrinogen degradation and in vitro plasminogen activation in plasma samples obtained during infusion of recombinant t-PA in patients with thromboembolic disease. Thus, MA-1C8 is a useful tool for discriminating between in vivo and in vitro fibrinolysis during thrombolytic therapy with t-PA.

An Essential Role of Factor VIII-Mediated Hemostasis in the Absence of von Willebrand Factor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3101-3101
Author(s):  
Yoshihiko Sakurai ◽  
Midori Shima ◽  
Shogo Kasuda ◽  
Shoko Omura ◽  
Masahiro Takeyama ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Essential Role ◽  
Bolus Administration ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background: The replacement therapy with plasma-derived factor VIII (FVIII)/von Willebrand factor (VWF) concentrates is the first line treatment for the patients with type 3 von Willebrand disease (VWD). However, development of anti-VWF alloantibodies (inhibitor) is a major problem since the inhibitor neutralizes the VWF activity and may cause anaphylactic reactions. As an alternative treatment, the usage of FVIII concentrates has been reported but the mechanism of the hemostatic effects remains to be elucidated. Objectives: The purpose of this study is to address the role of FVIII in the hemostatic mechanism in the absence of VWF by in vitro and ex vivo analysis in the treatment for type 3 VWD with recombinant FVIII (rFVIII). Patient/Methods: The patient is a 55-year-old male with type 3 VWD. Blood samples were obtained before and 30 min after bolus administration. Rotating thromboelastometry (ROTEM) assay was performed to examine global interactions in hemostasis. To elucidate the effect on platelet activation, α-thrombin- and shear-induced platelet aggregation studies were performed. Further, α-thrombin-induced [Ca2+]i rise was assessed using fura2-AM loaded platelets. Results and Implications: The patient underwent two surgical procedures of multiple teeth extractions successfully with minimal bleeding by bolus administration of rFVIII (50 IU/kg) before procedure and followed by continuous infusion at rate of 10 IU/kg/h for 15 hours. FVIII:C was elevated from 1.0% to 20~30% 30 min after bolus infusion and maintained ~15% after 12 h-continuous infusion. ROTEM analysis showed that infusion of rFVIII shortened clotting time (preinfusion 2083.8±784.3 sec vs. post-infusion 1022.0±191.5 sec) and clot formation time (pre 1267.3±455.4 sec vs. post 705.8±261.8 sec) and increased α (pre 8.5±7.4 degree vs. post 23.5±4.4 degree). The α value and CFT indicate the rate of increase of elastic shear modulus. Addition of rFVIII to preinfusion blood in vitro corrected ROTEM parameters and thrombin-induced aggregation dose-dependently. Infusion of FVIII enhanced thrombin-induced platelet aggregation (% maximal aggregation: pre 26.3% vs. post 98.2%) as well as low shear-induced platelet aggregation (% maximal aggregation: pre 18% vs. post 52%). Furthermore, infusion of rFVIII meliorated thrombin-induced intracellular calcium flux of washed platelets (thrombin 10 nM, Ca flux: pre 414.0 nM vs. post 620.6 nM). Recently, the cell-based model of hemostasis provides a solid foundation for the relation between platelet and coagulation. Although coagulation initiation occurs normally via the extrinsic pathway, amplification mediated by the intrinsic pathway is seriously disturbed in type 3 VWD due to the marked decrease in FVIII. Therefore, correction of FVIII could result in the improvement of hemostasis. Our data demonstrated the effectiveness of FVIII in the surgical treatment for type 3 VWD and further suggested that FVIII molecules are incorporated into platelet phospholipids to facilitate platelet activation as well as act directly to intrinsic pathways to normalize coagulation. Conclusions: Our observations suggested that FVIII plays an essential role in hemostasis in the absence of VWF and provided the rationale for the usage of rFVIII in the hemostatic management of type 3 VWD.

Sign in / Sign up

Export Citation Format

Share Document