γA/γ’ fibrinogen inhibits thrombin-induced platelet aggregation

2008 ◽  
Vol 100 (05) ◽  
pp. 837-846 ◽  
Author(s):  
Rehana S. Lovely ◽  
Chantelle M. Rein ◽  
Tara C. White ◽  
Sari A. Jouihan ◽  
Lynn K. Boshkov ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Active Site ◽  
Platelet Adhesion ◽  
Peptide Fragment ◽  
Flow Conditions ◽  
Amidolytic Activity ◽  
Direct Inhibition ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Inhibition Of Thrombin

SummaryThe minor γA/γ’ fibrinogen isoform contains a high affinity binding site for thrombin exosite II that is lacking in the major γA/γA fibrinogen isoform. We therefore investigated the biological consequences of the γ’ chain binding to thrombin. Thrombin-induced platelet aggregation was inhibited by γA/γ’ fibrinogen.Carboxyl terminal peptide fragment γ’410–427 from the γ’ chain was also inhibitory, with an IC50 of ∼200 µM in whole plasma. Deletion of the peptide from either the amino or carboxyl end significantly decreased inhibition. In contrast to thrombin-induced platelet aggregation, aggregation induced by epinephrine, ADP, arachidonic acid, or SFLLRN peptide showed little inhibition by the γ’ peptide. The inhibition of thrombin-induced platelet aggregation was not due to direct inhibition of the thrombin active site, since cleavage of a small peptidyl substrate was 91% of normal even in the presence of 1 mM γ’410–427.The γ’410–427 peptide blocked platelet adhesion to immobilized thrombin under both static and flow conditions,blocked soluble thrombin binding to platelet GPIbα, and inhibited PAR1 cleavage by thrombin. These results suggest that the γ’ chain of fibrinogen inhibits thrombin-induced platelet aggregation by binding to thrombin exosite II. Thrombin that is bound to the γ’ chain is thereby prevented from activating platelets, while retaining its amidolytic activity.

scholarly journals Thrombin Metz: characterization of the dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽  
1984 ◽  
Vol 63 (4) ◽  
pp. 927-934
Author(s):  
MJ Rabiet ◽  
M Jandrot-Perrus ◽  
JP Boissel ◽  
J Elion ◽  
F Josso
Keyword(s):  
High Performance ◽  
Fluorescence Enhancement ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Competitive Inhibitor ◽  
Amidolytic Activity ◽  
Direct Inhibition ◽  
High Affinity ◽  
Factor Va

Thrombin Metz and normal thrombin, resulting from activation of the respective prothrombins by factor Xa in the presence of calcium, phospholipid, and factor Va, were purified by chromatography on sulfopropyl Sephadex. By physicochemical criteria, thrombin Metz is identical to normal thrombin. Its functional properties were investigated in some reactions in which thrombin is classically involved. Thrombin Metz exhibits less than 4% of fibrinogen clotting activity. Both Km and Kcat, determined on S2238, are abnormal. Titration with the high-affinity competitive inhibitor of thrombin, DAPA, shows that fluorescence enhancement of the probe is only 34% in binding to thrombin Metz when compared to that observed in binding to normal thrombin. High-performance liquid chromatography has been used to measure the simultaneous rate of release of fibrinopeptides A and B. A decreased release rate for both fibrinopeptides, more marked for fibrinopeptide B, results in a slow fibrin polymerization, as followed by absorbance at 450 nm. Thrombin Metz is less than 5% as effective as normal thrombin in inducing platelet aggregation. Interaction with antithrombin III is slower than normal when followed by SDS gel electrophoresis and inhibition of the amidolytic activity of thrombin on S2238. This abnormality is not observed in the presence of heparin. However, thrombin Metz binds less tightly to a heparin-Sepharose column, and the direct inhibition of heparin on its activity on S2238 is weaker. From these results, we can predict that the defect in thrombin Metz affects the catalytic site or its vicinity and, jointly or consequently, the region of interaction of thrombin with antithrombin III and heparin.

Collagen Coated Gelatine Tubes for the Investigation of Platelet Adhesion and Subsequent Platelet Aggregation Under Controlled flow Conditions

1975 ◽  
Author(s):  
R. Muggli ◽  
H. R. Baumgartner
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Surface Coverage ◽  
Neutral Salt ◽  
Flow Conditions ◽  
Platelet Surface ◽  
Perfusion Chamber ◽  
Artificial Surface ◽  
Platelet Thrombi ◽  
Controlled Flow

Aggregometer studies do not discriminate between platelet adhesion and platelet aggregation. Therefore, we prepared a homogenous collagen surface which could be exposed to whole blood in a perfusion chamber under controlled flow conditions.Artificial “vessel” segments were prepared by dipping glass rods into 20% gelatine and, after air-drying, cross-linking the gelatine in 2.5% glutaraldehyde. Segments of 1 cm length were then drawn on the rod of the perfusion chamber and coated with 300 μl of neutral salt soluble collagen (2.2 mg/ml). Surface coverage with collagen was virtually complete (96%–100%).Uncoated or collagen-coated gelatine segments were exposed to citrated rabbit blood for periods up to 40 min. Platelet-surface interaction was evaluated morphometrically. On uncoated segments surface coverage with platelets amounted to 31% and 50% after 10 min and 40 min (the corresponding ratios of contact/spread platelets were 2.6 and 1.5). Only 0.1% thrombi were found. On collagen-coated segments surface coverage with platelets amounted to 57% and 83% after 10 min and 40 min (the corresponding ratios of contact/ spread platelets were 0.1 and 0.0); platelet thrombi were found on 33% and 42% of the surface after 10 min and 40 min.Platelet adhesion and subsequent aggregation on the collagen-coated artificial surface is similar to that observed on α-chymotrypsin digested subendothelium. The results suggest that fibrillar collagen triggers rapid spreading on a surface, a reaction which is closely associated with the formation of platelet thrombi. The latter phenomenon is thought to be caused by the release of aggregating agents from the spreading platelets.

Involvement of Activated Integrin α2β1 in the Firm Adhesion of Platelets onto a Surface of Immobilized Collagen under Flow Conditions

2000 ◽  
Vol 83 (05) ◽  
pp. 769-776 ◽  
Author(s):  
Ichiro Onitsuka ◽  
Tsutomu Imaizumi ◽  
Stephanie Jung ◽  
Masaaki Moroi
Keyword(s):  
Platelet Adhesion ◽  
Active Form ◽  
Tight Binding ◽  
Binding Property ◽  
Second Phase ◽  
Flow Conditions ◽  
Platelet Surface ◽  
High Affinity ◽  
Soluble Collagen ◽  
Induced Aggregation

SummaryRecently, we demonstrated that agonist-induced activation of the platelet surface collagen-receptor integrin α1 β2 converts it to an active form that can bind soluble collagen with high affinity (Jung, SM, Moroi, M: J Biol Chem 1998; 273: 14827-37). Here, the involvement of α2 β1 activation and the high affinity binding property of activated α2β1 in platelet adhesion to a collagen surface under flow conditions were analyzed. Platelet adhesion to immobilized collagen was measured in the presence of TS2/16, an activating anti-integrin α2β1 antibody, and inhibiting antibodies, Gi9 and 6F1. TS2/16 decreased the moving velocity of platelets on the collagen surface, but Gi9 and 6F1 increased it, indicating that α2β1 activation induces the tight binding of platelets to immobilized collagen under flow. Platelet adhesion, expressed as the surface area occupied by adhered platelets, in the presence of TS2/16 was similar to that in its absence. In contrast, adding Gi9 or 6F1 caused biphasic adhesion composed of a first phase, a lag phase whose length differed in each experiment, and a second phase adhesion with a rate similar to that of the control. This biphasic adhesion indicates that α2β1 activity is inhibited and also suggests that some other factor(s) may contribute to the adhesion under flow. At concentrations where neither 6F1 nor Gi9 affected collagen-induced aggregation, these antibodies inhibited soluble collagen binding to thrombin-activated platelets. Only at much higher concentration did 6F1 inhibit collagen-induced aggregation. TS2/16 had no effect on the aggregation. The present results are evidence against the major involvement of integrin α2β1 in platelet aggregation; instead, they indicate that integrin α2β1 would be mainly associated with the tight binding of platelets to collagen.

Magnesium maintains endothelial integrity, up-regulates proteolysis of ultra-large von Willebrand factor, and reduces platelet aggregation under flow conditions

2008 ◽  
Vol 99 (03) ◽  
pp. 586-593 ◽  
Author(s):  
Miguel Cruz ◽  
Khatira Aboulfatova ◽  
Cecilia Martin ◽  
Hiuwan Choi ◽  
Angela Bergeron ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Human Umbilical Cord ◽  
Flow Conditions ◽  
Endothelial Functions ◽  
Endothelial Integrity ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Insight Into

SummaryMg ++ regulates endothelial functions and has anti-inflammatory effects. Its effects on thrombosis have been demonstrated, but the mechanism remains poorly understood.We investigated the roles of MgSO4 in regulating the release and cleavage of the prothrombotic ultra-large (UL) von Willebrand factor (VWF) and VWF-mediated platelet adhesion and aggregation.Washed platelets were perfused over cultured endothelial cells from human umbilical cord veins under a shear stress of 2.5 dyn/cm2. Release and cleavage of ULVWF by ADAMTS-13 was measured in the absence or presence of physiological or therapeutic levels of MgSO4. Whole blood or plasma-free reconstituted blood was perfused over immobilized collagen to measure the effect of MgSO4 on platelet adhesion and aggregation. Also studied were the effects of MgSO4 on ristocetin-induced platelet aggregation andVWF-collagen interaction.Maintenance of endothelial integrity required physiological levels of MgSO4, but exogenous MgSO4 showed no additional benefits.Exogenous MgSO4 significantly enhanced the cleavage of the newly released ULVWF strings by ADAMTS-13 and markedly reduced platelet aggregation on immobilized collagen under flow conditions.This effect is likely to be mediated through VWF as Mg++ partially inhibited ristocetin-induced platelet aggregation andVWF binding to collagen.MgSO4 is critical for maintaining endothelial integrity and regulates ULVWF proteolysis and aggregation under flow conditions. These results provide a new insight into additional mechanisms involved with magnesium therapy.

scholarly journals Thrombin Metz: characterization of the dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽  
1984 ◽  
Vol 63 (4) ◽  
pp. 927-934 ◽  
Author(s):  
MJ Rabiet ◽  
M Jandrot-Perrus ◽  
JP Boissel ◽  
J Elion ◽  
F Josso
Keyword(s):  
High Performance ◽  
Fluorescence Enhancement ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Competitive Inhibitor ◽  
Amidolytic Activity ◽  
Direct Inhibition ◽  
High Affinity ◽  
Factor Va

Abstract Thrombin Metz and normal thrombin, resulting from activation of the respective prothrombins by factor Xa in the presence of calcium, phospholipid, and factor Va, were purified by chromatography on sulfopropyl Sephadex. By physicochemical criteria, thrombin Metz is identical to normal thrombin. Its functional properties were investigated in some reactions in which thrombin is classically involved. Thrombin Metz exhibits less than 4% of fibrinogen clotting activity. Both Km and Kcat, determined on S2238, are abnormal. Titration with the high-affinity competitive inhibitor of thrombin, DAPA, shows that fluorescence enhancement of the probe is only 34% in binding to thrombin Metz when compared to that observed in binding to normal thrombin. High-performance liquid chromatography has been used to measure the simultaneous rate of release of fibrinopeptides A and B. A decreased release rate for both fibrinopeptides, more marked for fibrinopeptide B, results in a slow fibrin polymerization, as followed by absorbance at 450 nm. Thrombin Metz is less than 5% as effective as normal thrombin in inducing platelet aggregation. Interaction with antithrombin III is slower than normal when followed by SDS gel electrophoresis and inhibition of the amidolytic activity of thrombin on S2238. This abnormality is not observed in the presence of heparin. However, thrombin Metz binds less tightly to a heparin-Sepharose column, and the direct inhibition of heparin on its activity on S2238 is weaker. From these results, we can predict that the defect in thrombin Metz affects the catalytic site or its vicinity and, jointly or consequently, the region of interaction of thrombin with antithrombin III and heparin.

Platelet cGMP, but not cAMP, inhibits thrombin-induced platelet adhesion to pulmonary vascular endothelium

1992 ◽  
Vol 263 (2) ◽  
pp. H606-H612
Author(s):  
C. M. Venturini ◽  
L. K. Weston ◽  
J. E. Kaplan
Keyword(s):  
Nitric Oxide ◽  
Platelet Aggregation ◽  
Vascular Endothelium ◽  
Platelet Adhesion ◽  
Flow Conditions ◽  
Cyclic Monophosphate ◽  
Inhibition Of Platelet Aggregation ◽  
Endothelial Monolayers ◽  
Platelet Aggregate ◽  
Pulmonary Vascular Endothelium

We have compared the effects of intracellular pathways initiated by nitric oxide and prostacyclin on thrombin-induced platelet adhesion to endothelial cells. Platelet aggregate adhesion was enhanced when endothelial monolayers were pretreated with NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide production. In addition, decreased platelet aggregate adhesion was seen when platelets were pretreated with 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP). Single platelet adhesion in isolated perfused lungs under flow conditions in the presence of shear was also assessed. Pretreatment of platelets with either Iloprost, in a dose sufficient to decrease platelet aggregation, or 8-bromo-cAMP did not affect platelet adhesion. However, pretreatment of platelets with 8-bromo-cGMP significantly reduced single platelet adhesion to endothelium. These studies illustrate that nitric oxide inhibits platelet adhesion to endothelium in the presence of shear. They further indicate that prostacyclin is also a regulator of this response but has effects more specifically related to the inhibition of platelet aggregation than platelet-endothelium interactions.

Increased platelet adhesion under flow conditions is induced by both thalassemic platelets and red blood cells

2008 ◽  
Vol 100 (05) ◽  
pp. 864-870 ◽  
Author(s):  
Alexander Brill ◽  
Orly Zelig ◽  
Ada Goldfarb ◽  
Eliezer Rachmilewitz ◽  
Neta Goldschmidt ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Lipid Membrane ◽  
Platelet Rich Plasma ◽  
Blood Type ◽  
Flow Conditions ◽  
Normal Platelet ◽  
History Of ◽  
Average Size ◽  
Risk For Thrombosis

SummaryThromboembolic complications are not uncommon in thalassemia. Previous studies suggest increased platelet aggregation and a potential role of pathological changes in the red blood cell (RBC) lipid membrane, induced by oxidative stress. In the present study,platelet adhesion and the effect of thalassemic RBC on platelet adhesion under flow conditions were evaluated, using the Cone and Plate (let)Analyzer(CPA).Twenty-two β-thalasse-mia patients and 22 blood type-matched healthy controls were studied. An increased platelet adhesion (% surface coverage, SC),was observed in patients as compared to controls (p<0.05). When platelet count and haematocrit were normalized by auto-logous reconstitution, a significant increase in platelet aggregation (average size,AS) was observed (p<0.05). Increased platelet adhesion (SC and AS), was demonstrated in six patients with a history of thrombosis as compared to 16 patients without any history of thrombosis (p≤0.007) and in 17 splenectomized patients as compared to five non-splenectomized patients (p=0.003). In reconstitution studies, thalassemic RBC mixed with normal platelet-rich plasma significantly increased platelet adhesion compared to normal RBC (SC p<0.03, AS p<0.02). Thalassemic platelets reconstituted with normal RBC, had increased aggregation (AS, p<0.004) in comparison with normal platelets.The results indicate that increased platelet adhesion in β-thalassemia is induced by both platelets and RBC. Increased platelet adhesion correlated with clinical thrombotic events and thus may suggest a mechanism of thrombosis in thalassemic patients.The potential application of the CPA in identifying thalassemic patients with high risk for thrombosis should be studied prospectively in a larger cohort of patients.

A G-quartet oligonucleotide blocks glycoprotein Ib-mediated platelet adhesion and aggregation under flow conditions

2009 ◽  
Vol 102 (09) ◽  
pp. 529-537 ◽  
Author(s):  
Zhou Zhou ◽  
Aubrey Bernardo ◽  
Qiqing Zhu ◽  
Yongli Guan ◽  
Wensheng Sun ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Flow Conditions ◽  
Β Sheets ◽  
A1 Domain ◽  
Von Willebrand ◽  
Potential Interactions ◽  
Willebrand Factor ◽  
Binding Subunit

SummaryPlatelets arrest bleeding by adhering to and aggregating on the subendothelium exposed at the site of vessel injury.This process is initiated by the interaction between the subendothelium von Willebrand factor (VWF) and the glycoprotein (GP) Ib-IX-V complex on platelets.However,the same interaction also results in thrombosis at the site of a ruptured atherosclerotic plaque. Reagents regulating the GP Ib-VWF interaction will therefore have direct impact on haemostasis and thrombosis. We have characterised an oligonucleotide G-quartet (T30923) that specifically blocks VWF binding to GP Ibα, theVWF-binding subunit of the GP Ib-IX-V complex.We evaluated the potential interactions of T30923 with GP Ibα and VWF A1 domain by computer simulated molecular dockings, which identified four T30923 docking sites in the β-sheets of the N-terminal region of GP Ibα (E14-D18, S39, D63-S64, and D83-S85). Experimentally,T30923 bound GP Ibα and dose-dependently blocked platelet aggregation induced by ristocetin and thrombin, but not by botrocetin, collagen, TRAP, and ADP. It also blocked shear-induced platelet aggregation and thrombus formation on immobilized VWF under arterial shear stress. These results demonstrate that T30923 may have therapeutic potentials to regulate the GP IbαVWF interaction.

scholarly journals von Willebrand factor (VWF) propeptide binding to VWF D′D3 domain attenuates platelet activation and adhesion

Blood ◽  
2012 ◽  
Vol 119 (20) ◽  
pp. 4769-4778 ◽  
Author(s):  
Sri R. Madabhushi ◽  
Chengwei Shang ◽  
Kannayakanahalli M. Dayananda ◽  
Kate Rittenhouse-Olson ◽  
Mary Murphy ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Flow Chamber ◽  
High Affinity ◽  
Intracellular Compartments ◽  
Storage Granules ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Kinetics Of

Abstract Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D′D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D′D3 domain. At pH 6.2 and 10mM Ca2+, conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (KD = 0.2nM, koff = 8 × 10−5 s−1). Significant, albeit weaker, binding (KD = 25nM, koff = 4 × 10−3 s−1) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca2+. This interaction was also observed in human plasma (KD = 50nM). The addition of recombinant VWFpp in both flow-chamber–based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D′D3 mAb DD3.1, which blocks VWFpp binding to VWF-D′D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.

Fine Structure of Platelet Interaction with a New Microcrystalline Collagen Preparation

1974 ◽  
Vol 32 ◽  
pp. 58-59
Author(s):  
W. H. Zucker ◽  
R. G. Mason
Keyword(s):  
Fine Structure ◽  
Platelet Aggregation ◽  
Hydrochloric Acid ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Hemostatic Agent ◽  
Native Collagen ◽  
Fibrillar Morphology ◽  
Clinical Interest ◽  
New Form

Platelet adhesion initiates platelet aggregation and is an important component of the hemostatic process. Since the development of a new form of collagen as a topical hemostatic agent is of both basic and clinical interest, an ultrastructural and hematologic study of the interaction of platelets with the microcrystalline collagen preparation was undertaken.In this study, whole blood anticoagulated with EDTA was used in order to inhibit aggregation and permit study of platelet adhesion to collagen as an isolated event. The microcrystalline collagen was prepared from bovine dermal corium; milling was with sharp blades. The preparation consists of partial hydrochloric acid amine collagen salts and retains much of the fibrillar morphology of native collagen.

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