Platelet microparticle formation and thrombin generation under high shear are effectively suppressed by a monoclonal antibody against GPIbα

2006 ◽  
Vol 96 (12) ◽  
pp. 774-780 ◽  
Author(s):  
Luca Pontiggia ◽  
Beat Steiner ◽  
Hans Ulrichts ◽  
Hans Deckmyn ◽  
Marc Forestier ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Annexin V ◽  
High Shear ◽  
Double Labeling ◽  
Platelet Aggregometry ◽  
Microparticle Formation ◽  
Arterial Stenoses ◽  
Von Willebrand

SummaryWe studied the inhibition of platelet microparticle (MP) formation and thrombin generation under high shear forces. We hypothesized that an inhibitor of the GPIbα-von Willebrand factor (vWF) interaction would be more effective in suppressing MP formation and thrombin generation than GPIIb/IIIa inhibitors. Platelet-rich plasma (PRP) anticoagulated with PPACK (D-Phe-Pro-Arg chloromethyl ketone) was exposed in a coneand-plate viscometer (shear: 5,000 s−1 for5 min) in the presence of antagonists to GPIbα (the monoclonal antibody [Mab] Ib-23) or to GPIIb/IIIa (abciximab, tirofiban, eptifibatide) at their IC90 determined in platelet aggregometry with ristocetin or ADP, respectively. We used double labeling (CD41-PE and annexin-V-FITC) for flow cytometric detection of MP and their aminophospholipid exposure. Thrombin generation was measured using PRP prepared fromACD anticoagulated blood. About 40% of the thrombin generation was found to be mediated by the MP fraction of the PRP. Blockade of GPIbα with Mab Ib-23 reduced MP formation and thrombin generation by 50%, and was more effective than any GPIIb/IIIa antagonist. The combination of Mab Ib-23 with one of the GPIIb/IIIa inhibitors further reduced the MP formation to ∼30%. The antibody also partially inhibited thrombin induced platelet aggregation. Epitope mapping suggested that Mab Ib-23 binds between the amino acids 201 and 268 of GPIbα, explaining the interference with vWF and thrombin interaction. In contrast to the commonly used GPIIb/IIIa antagonists, the blockade of GPIbα with Mab Ib-23 effectively reduces the prothrombotic MP generation and thrombin formation at shear rates typically found in arterial stenoses.

Circulating platelet-derived microparticles in systemic lupus erythematosus

2006 ◽  
Vol 95 (01) ◽  
pp. 94-99 ◽  
Author(s):  
Gino Alfaro ◽  
Manuela Goycoolea ◽  
Teresa Quiroga ◽  
Mauricio Ocqueteau ◽  
Loreto Massardo ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Lupus Erythematosus ◽  
Thrombin Generation ◽  
Thrombus Formation ◽  
Annexin V ◽  
Coagulation System ◽  
Systemic Lupus ◽  
Double Labeling ◽  
Cellular Source ◽  
Risk For Thrombosis

SummaryThe risk for thrombosis is significantly increased in systemic lupus erythematosus (SLE), affecting both venous and arterial vessels. Activated platelets are known to participate in thrombus formation and growth. A general feature of activated cells is the shedding of microparticles (MP) which support coagulation by exposure of negatively charged phospholipids and possibly tissue factor (TF). In this work we characterized circulating MP in patients with SLE and their relationship with a procoagulant state. Thirty patients with SLE (aged 15–72 years, mean age 38 years) and 20 healthy controls (aged 22–54 years, mean age 34 years) were studied; patients fulfilled 4 revised criteria for SLE. The number and cellular source of circulating MP were determined by flow cytometry using double labeling with specific monoclonal antibodies and annexin V. Thrombin generation was measured as the endogenous thrombin potential (ETP) without the addition of exogenous phospholipids and TF; under these conditions the generation of thrombin depended directly on the number of MP present in plasma. Thrombin anti-thrombin (TAT) and plasmin-antiplasmin (PAP) complexes were measured by ELISA. Compared to the controls, circulating MP were significantly elevated in the patient group (1218 ± 136 vs 653 ± 74 x103/ml plasma, p: 0.0007). In both groups, most of these MP were of platelet origin (927± 131 vs 517 ± 72 x103/ml plasma, p:0.009 ). ETP was higher among patients as compared to the controls (804± 64 vs 631 ± 37 nM thrombin, p: 0.025). Plasma levels of TAT in patients and controls were 3.4 ± 0.8 and 1.4 ± 0.5 µg/L, respectively (p:0.04), and of PAP complexes were 62.5 ± 14 and 24.05± 2.5 µg/ml, respectively (p: 0.014).The number of platelet-derived MP correlated significantly with thrombin generation (r: 0.42; p: 0.038) and TAT levels (r: 0.40; p: 0.035).We did not find an association of circulating MP with disease activity nor with the presence of antiphospholipid antibodies. The increased number of circulating platelet-derived microparticles and their association with high ETP and activation of the coagulation system suggest that these microparticles play an important role in the pathogenesis of the prothrombotic state in SLE patients.

scholarly journals Unique interactions of asialo von Willebrand factor with platelets in platelet-type von Willebrand disease

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1804-1809 ◽  
Author(s):  
JL Miller ◽  
ZM Ruggeri ◽  
VA Lyle
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Specific Binding ◽  
Platelet Rich Plasma ◽  
Von Willebrand Disease ◽  
Normal Platelet ◽  
High Affinity Binding Site ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Formalin Fixed

Abstract The present studies demonstrate that platelets from patients with platelet-type von Willebrand disease show specific and saturable binding of asialo von Willebrand factor (AS-vWF) under conditions where such binding is not observed with normal platelets. Although specific binding of 125I-AS-vWF to formalin-fixed normal platelets could not be demonstrated, specific binding to fixed patient platelets was seen with an apparent Kd of 1.3 micrograms/mL and specific maximally bound ligand of 0.40 micrograms/10(8) platelets. Preincubation of patient platelets with the antiglycoprotein Ib (anti-GPIb) monoclonal antibody AS-2 reduced total binding close to the level of computer-estimated nonspecific binding. In contrast, binding was not reduced by preincubation with anti-GPIIb/IIIa monoclonal antibody or with 5 mmol/L EDTA. Under stirring conditions, the binding of AS-vWF to fixed patient platelets was accompanied by a strong agglutination response. AS-vWF- induced agglutination was similarly observed in patient but not normal platelet-rich plasma (PRP) in the presence of 5 mmol/L EDTA. In the absence of EDTA, AS-vWF produced a full aggregation response in patient PRP at concentrations as low as 0.1 microgram/mL in contrast to the 2 to 20 micrograms/mL required by normal PRP. Both thromboxane B2 formation and adenosine triphosphate secretion showed an AS-vWF concentration dependence paralleling the aggregation responses. These studies show that a major difference in the platelets from patients with platelet-type von Willebrand disease is the presence of an exposed, high-affinity binding site associated with GPIb that recognizes AS-vWF.

scholarly journals Plasminogen interactions with platelets in plasma

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1530-1535 ◽  
Author(s):  
B Adelman ◽  
A Rizk ◽  
E Hanners
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Platelet Rich Plasma ◽  
Aminocaproic Acid ◽  
Binding Domains ◽  
Fibrinogen Binding ◽  
Platelet Binding ◽  
Epsilon Aminocaproic Acid ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In this report we used a fluorescent flow cytometry-based assay to examine plasminogen binding to platelets in plasma. Our data indicate that platelets activated in platelet-rich plasma (PRP) by adenosine-5′- diphosphate (ADP) or thrombin bind plasminogen to their surface. Fab fragments of the monoclonal antibody LJ-CP8 that are directed against the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex inhibit both plasminogen and fibrinogen binding to ADP-stimulated platelets as does 5 mmol/L EDTA. Platelet aggregation and plasminogen and fibrinogen binding are also concurrently inhibited by the Gly-Arg- Asp (RGD) analogue Gly-Arg-Gly-Asp-Ser (GRGDS) when it is added to PRP before ADP stimulation. The scrambled peptide analogue SDGRG has no effect. The monoclonal antibody 6D1, directed against the von Willebrand factor binding site on GPIb, has no effect on plasminogen- platelet binding, nor does antithrombospondin antibody. epsilon- Aminocaproic acid (EACA), however, inhibits plasminogen binding to ADP- activated platelets. These data indicate that plasminogen binds to platelets activated in plasma, that binding occurs on platelet GPIIb/IIIa, and that binding may be mediated via plasminogen association with fibrinogen via lysine binding domains. Finally, we found both plasminogen and fibrinogen on resting platelets in PRP and demonstrated that they are equally displaced by EDTA, LJ-CP8, and 10E5 (an additional anti-GPIIb/IIIa monoclonal antibody). Plasminogen is also equally displaced by EACA. These data suggest that plasminogen is also bound to GPIIb/IIIa on resting platelets, possibly also via interaction with fibrinogen.

scholarly journals von Willebrand factor binding to myosin assists in coagulation

2020 ◽  
Vol 4 (1) ◽  
pp. 174-180 ◽  
Author(s):  
Veronica H. Flood ◽  
Tricia L. Slobodianuk ◽  
Daniel Keesler ◽  
Hannah K. Lohmeier ◽  
Scot Fahs ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Factor V ◽  
Factor X ◽  
Skeletal Muscle Myosin ◽  
Myosin Binding ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (VWF) binds to platelets and collagen as a means of facilitating coagulation at sites of injury. Recent evidence has shown that myosin can serve as a surface for thrombin generation and binds to activated factor V and factor X. We studied whether VWF can also bind myosin as a means of bringing factor VIII (FVIII) to sites of clot formation. A myosin-binding assay was developed using skeletal muscle myosin to measure VWF binding, and plasma-derived and recombinant VWF containing molecular disruptions at key VWF sites were tested. Competition assays were performed using anti-VWF antibodies. FVIII binding to myosin was measured using a chromogenic FVIII substrate. Thrombin generation was measured using a fluorogenic substrate with and without myosin. Wild-type recombinant VWF and human plasma VWF from healthy controls bound myosin, whereas plasma lacking VWF exhibited no detectable myosin binding. Binding was multimer dependent and blocked by anti-VWF A1 domain antibodies or A1 domain VWF variants. The specific residues involved in myosin binding were similar, but not identical, to those required for collagen IV binding. FVIII did not bind myosin directly, but FVIII activity was detected when VWF and FVIII were bound to myosin. Myosin enhanced thrombin generation in platelet-poor plasma, although no difference was detected with the addition of myosin to platelet-rich plasma. Myosin may help to facilitate delivery of FVIII to sites of injury and indirectly accelerate thrombin generation by providing a surface for VWF binding in the setting of trauma and myosin exposure.

Comparison of Three Different Platelet Function Assays to Determine Aspirin Sensitivity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3901-3901
Author(s):  
Rachelle Blain ◽  
Peggy Nakagawa ◽  
Thomas Kunicki ◽  
Melissa Belvedere ◽  
Diane Nugent
Keyword(s):  
Platelet Function ◽  
Whole Blood ◽  
Large Scale ◽  
Classical Method ◽  
Platelet Rich Plasma ◽  
High Shear ◽  
Aspirin Sensitivity ◽  
Normal Individuals ◽  
Platelet Aggregometry ◽  
Coagulation Assay

Abstract In large studies of patients with cardiac disease, it was observed that up to 9% are ASA resistant (ASAR) and 23% are ASA semi responders using the platelet function analyzer (PFA-100), which measures whole blood platelet adhesion and aggregation under high shear. The prevalence of ASAR is of great clinical importance. With an estimated 20 million patients in the USA taking ASA for prevention of atherosclerotic events, even a 10% incidence equates to more than two million patients receiving inadequate anti-platelet therapy. Given the widespread use of ASA, more reliable and rapid methods to measure aspirin sensitivity are needed. Importantly, future large scale studies to determine the effect of monitoring ASA sensitivity to optimize therapy are compromised due to current lack of uniformity in assessing ASAR from center to center. The classical method of platelet aggregometry is labor-intensive and not readily adaptable to the clinical setting. Moreover, platelet aggregometry measures platelet responses under low shear, which does not simulate the high shear conditions expected to be involved in arterial thrombosis. In contrast, the PFA-100 does measure thrombus formation under high shear. The new TEG platelet coagulation assay uses whole blood, includes high shear, and is a point of care method. We compared these three techniques to assess aspirin effects on platelet function and clot formation: PFA-100 with collagen-epinephrine cartridges, TEG platelet coagulation assay, and standard optical aggregation in platelet rich plasma using arachidonic acid (AA) and adenosine diphosphate (ADP) as agonists. RESULTS: We found significant differences between the PFA-100, TEG platelet function, and standard optical aggregation. Twelve normal individuals previously defined as ASA sensitive or ASAR, using the PFA-100, were studied at baseline and following three days of oral aspirin at 81mg/day. We found that all four patients determined to be ASAR by PFA-100 were found to be sensitive in TEG and aggregometer assays when using AA as the agonist. Furthermore, some participants showed a gain of platelet function following ASA when studied on the TEG and aggregometer using ADP as the agonist. Currently, it is unclear which response to ASA treatment is most important to predict cardiovascular complications in normal individuals or patients placed on aspirin. Gum et al, showed that increased urinary secrection of thromboxane metabolites, suggesting ASA insensitivity, is associated with a higher incidence of cardiovascular events. Unfortunately, this test may simply indicate poor patient compliance rather than true ASAR, so a clear demonstration of platelet sensitivity will also be necessary. Our data confirms the need for a collaborative trial comparing different platelet assays to assess true ASA sensitivity together with concurrent measurements of urinary thromboxane metabolite levels. Knowing which assay best links aspirin sensitivity to disease outcome will allow physicians to better manage normal individuals and patients at risk for significant cardiovascular disease.

Effects of von Willebrand factor concentration and platelet collision on shear-induced platelet activation

2008 ◽  
Vol 100 (07) ◽  
pp. 60-68 ◽  
Author(s):  
Zhenyue Gao ◽  
Fang Liu ◽  
Ziqiang Yu ◽  
Xia Bai ◽  
Fengyuan Zhuang ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Annexin V ◽  
Flow Chamber ◽  
Platelet Glycoprotein ◽  
High Shear ◽  
Shear Rates ◽  
Von Willebrand ◽  
The Impact ◽  
Willebrand Factor

SummaryThe binding of plasma von Willebrand factor (vWF) to platelet glycoprotein (GP) Ibα in a high shear stress field, and subsequent integrin-GPIIb/IIIa-vWF conjunction induces platelet aggregation (SIPA). However, the specific biomechanical mechanism of the vWF-GPIb interaction still remains to be elucidated. A parallel-plate rectangular flow chamber was built to simulate a stenopeic artery flow pattern. Using the flow chamber, we examined shear- induced platelet activation (SIPAct) at different vWF concentrations (5–25 µg/ml) and several simulated stenotic high shear rates. P-selectin expression on the platelets and annexin V binding to the platelets were used as two markers of platelet activation. At different localized shear rates (3,000 s-1–9,500 s-1), the percentage of annexin V and P-selectin positive cells increased from 8.3 ± 0.4% to 22.3 ± 1.8% ( p 0.05) and from 17.4 ± 0.5% to 33.5 ± 2.5% (p 0.05),respectively. As the vWF concentration increased from 5 µg/ml to 25 µg/ml, the annexinV binding rate increased from 7.2 ± 0.6% to 53.4 ± 3.8% (p 0.05), and P-selectin expression increased from 16.5 ± 1.2% to 65.9 ± 5.2% (p 0.05). A test in a uniform shear field using cone-plate viscometer rheometry showed that the platelet activation rate was proportional to the platelet concentration. This result suggests that platelet collision is one of the impact factors of SIPAct.

scholarly journals Plasminogen interactions with platelets in plasma

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1530-1535
Author(s):  
B Adelman ◽  
A Rizk ◽  
E Hanners
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Platelet Rich Plasma ◽  
Aminocaproic Acid ◽  
Binding Domains ◽  
Fibrinogen Binding ◽  
Platelet Binding ◽  
Epsilon Aminocaproic Acid ◽  
Von Willebrand ◽  
Willebrand Factor

In this report we used a fluorescent flow cytometry-based assay to examine plasminogen binding to platelets in plasma. Our data indicate that platelets activated in platelet-rich plasma (PRP) by adenosine-5′- diphosphate (ADP) or thrombin bind plasminogen to their surface. Fab fragments of the monoclonal antibody LJ-CP8 that are directed against the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex inhibit both plasminogen and fibrinogen binding to ADP-stimulated platelets as does 5 mmol/L EDTA. Platelet aggregation and plasminogen and fibrinogen binding are also concurrently inhibited by the Gly-Arg- Asp (RGD) analogue Gly-Arg-Gly-Asp-Ser (GRGDS) when it is added to PRP before ADP stimulation. The scrambled peptide analogue SDGRG has no effect. The monoclonal antibody 6D1, directed against the von Willebrand factor binding site on GPIb, has no effect on plasminogen- platelet binding, nor does antithrombospondin antibody. epsilon- Aminocaproic acid (EACA), however, inhibits plasminogen binding to ADP- activated platelets. These data indicate that plasminogen binds to platelets activated in plasma, that binding occurs on platelet GPIIb/IIIa, and that binding may be mediated via plasminogen association with fibrinogen via lysine binding domains. Finally, we found both plasminogen and fibrinogen on resting platelets in PRP and demonstrated that they are equally displaced by EDTA, LJ-CP8, and 10E5 (an additional anti-GPIIb/IIIa monoclonal antibody). Plasminogen is also equally displaced by EACA. These data suggest that plasminogen is also bound to GPIIb/IIIa on resting platelets, possibly also via interaction with fibrinogen.

scholarly journals The Effect of DDAVP Infusion on Thrombin Generation in Platelet-rich Plasma of von Willebrand Type 1 and in Mild Haemophilia A Patients

2000 ◽  
Vol 84 (10) ◽  
pp. 638-642 ◽  
Author(s):  
Irene Keularts ◽  
K. Hamulyak ◽  
H.C. Hemker ◽  
Suzette Béguin
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Von Willebrand Disease ◽  
Clotting Factor ◽  
Haemophilia A ◽  
The Individual ◽  
Von Willebrand ◽  
Mild Haemophilia

SummaryIn von Willebrand disease (vWD) type 1 and mild haemophilia A patients we studied the effect of an infusion of DDAVP (0.3 µg/kg body weight) on thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Baseline thrombin generation in PRP was diminished both in the haemophilia A and vWD patients. It was normal in vWD plasma when sufficient procoagulant phospholipids were present, either via adding phospholipid vesicles to PPP or via scrambling of the platelet membrane with ionomycin in PRP. In haemophilia A plasma, thrombin generation did not normalize by providing procoagulant phospholipids. Treatment with DDAVP temporarily restored thrombin generation in PRP to normal in both diseases.To investigate the individual roles of von Willebrand factor (vWF) and factor VIII, we also studied the effect of factor VIII infusion on thrombin generation in a severe haemophilia patient. It appears that at a fixed normal vWF concentration, <25% factor VIII is sufficient for normal thrombin generation in PRP. At a sufficient factor VIII concentration, however, thrombin generation is still lower than normal in vWD patients; ∼40% of vWF is required for half-normal thrombin generation in PRP.It thus appears that vWF is also a clotting factor, in the sense that it is required for normal thrombin generation. This underlines the importance of the interaction between coagulation and the platelets in normal haemostasis. Thrombin generation in PRP appears to be a suitable test to reflect the combined function.

scholarly journals Fibrin-Dependent Platelet Procoagulant Activity Requires GPIb Receptors and von Willebrand Factor

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 564-570 ◽  
Author(s):  
S. Béguin ◽  
R. Kumar ◽  
I. Keularts ◽  
U. Seligsohn ◽  
B.S. Coller ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Von Willebrand Disease ◽  
Procoagulant Activity ◽  
Increased Risk ◽  
Coagulation Proteins ◽  
A Minor ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Thrombin generation in platelet-rich plasma (PRP) involves complex interactions between platelets and coagulation proteins. We previously reported that the addition of fibrin to PRP enhances tissue-factor initiated thrombin generation by ≈ 40%, and the current studies were designed to assess the mechanism(s) underlying thrombin generation in the absence and presence of fibrin. Blocking platelet GPIIb/IIIa + vβ3 receptors with a monoclonal antibody (MoAb) inhibited basal thrombin generation, but did not affect the enhancement produced by fibrin. In contrast, blocking GPIb with any of three different MoAbs had no effect on basal thrombin generation, but essentially eliminated fibrin enhancement of thrombin generation. When thrombin generation was tested in PRP deficient in von Willebrand factor (vWF), both basal and fibrin-enhanced thrombin generation were markedly reduced, and the addition of factor VIII did not normalize thrombin generation. Botrocetin, which induces the binding of vWF to GPIb, enhanced thrombin generation. In all studies, the ability of PRP to support thrombin generation correlated with the production of platelet-derived microparticles and serum platelet-derived procoagulant activity. Thus, two separate mechanisms, both of which depend on vWF, appear to contribute to platelet-derived procoagulant activity: one is independent of fibrin and relies primarily on GPIIb/IIIa, but with a minor contribution from vβ3; and the other is fibrin-dependent and relies on GPIb. These data may have implications for understanding the mechanisms of the abnormalities in serum prothrombin times reported in Bernard-Soulier syndrome, hemorrhage in von Willebrand disease (vWD), and the increased risk of thrombosis associated with elevated vWF levels.

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