The Effect of Platelet PlA Polymorphism on Experimental Thrombus Formation in Man Depends on Blood Flow and Thrombogenic Substrate

2001 ◽  
Vol 85 (06) ◽  
pp. 1097-1103 ◽  
Author(s):  
Kjell Sakariassen ◽  
Hélène Grandjean ◽  
Claire Thalamas ◽  
Bernard Boneu ◽  
Pierre Sié ◽  
...  
Keyword(s):  
Blood Flow ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Flow Properties ◽  
Shear Rates ◽  
Perfusion Chamber ◽  
Platelet Receptor ◽  
Platelet Deposition ◽  
Coronary Syndromes ◽  
Platelet Thrombus

SummaryA number of studies have reported conflicting data on the association of the PlA1/PlA2 polymorphism of the GPIIIa gene and coronary syndromes. We have investigated the effect of this polymorphism on experimental platelet thrombus formation in man. Forty healthy male volunteers were genotyped for the PlA1/PlA2 polymorphism. Thrombus formation was induced ex vivo by exposing a tissue factor (TF) or a collagencoated coverslip in a parallel plate perfusion chamber to native blood for 2 and 4 min. The shear rates at these surfaces were 650 and 2,600 s–1. Platelet and fibrin deposition was quantified by immunoenzymatic methods. The frequencies of PlA1/PlA1 and PlA1/PlA2 genotypes were 52.5% and 47.5%, respectively. Ex vivo deposition of fibrin on TF was not affected by the PlA1/PlA2 polymorphism. However, the ex vivo platelet deposition at 650 s–1 was higher in blood from PlA1/PlA1 individuals than in PlA1/PlA2 individuals (P = 0.008 at 4 min). On collagen, neither fibrin nor platelet deposition was significantly affected by the PlA1/PlA2 polymorphism. Platelet thrombus formation is significantly influenced by genetic variations in the GPIIIa platelet receptor. This effect depends on the blood flow properties and the nature of the thrombogenic stimulus.

Role of 4 platelet membrane glycoprotein polymorphisms on experimental arterial thrombus formation in men

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 3159-3161 ◽  
Author(s):  
Yves Cadroy ◽  
Kjell S. Sakariassen ◽  
Jean-Paul Charlet ◽  
Claire Thalamas ◽  
Bernard Boneu ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Blood Flow Rate ◽  
Variable Number ◽  
Kozak Sequence ◽  
Shear Rates ◽  
Perfusion Chamber ◽  
Arterial Thrombus ◽  
Platelet Thrombus

Abstract This study investigates whether the polymorphisms of 3 important platelet receptors affected experimental thrombus formation in men. Forty healthy male volunteers randomly recruited were genotyped for the variable number of tandem repeat (VNTR) of GPIbα, the −5T/C polymorphism in the Kozak sequence of GPIbα, the 807C/T polymorphism of GPIa, and the PlA1/PlA2polymorphism of GPIIb/IIIa. Platelet thrombus formation was induced ex vivo by exposing a collagen-coated coverslip in a parallel plate perfusion chamber to native blood for 4 minutes. The shear rates at the collagen surface were 650 and 2600 s−1. At 2600 s−1 platelet thrombus formation was significantly related only to the 807C/T polymorphism. In contrast, at 650 s−1thrombus formation was significantly altered only by the Kozak sequence polymorphism. The VNTR and the PlA1/PlA2polymorphisms did not influence thrombus formation. Thus, platelet thrombus formation is significantly influenced by genetic variations of the GPIbα and GPIa receptors. The effect of these polymorphisms was dependent on the blood flow rate.

scholarly journals A novel nucleotide-based thrombin inhibitor inhibits clot-bound thrombin and reduces arterial platelet thrombus formation

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 677-682 ◽  
Author(s):  
WX Li ◽  
AV Kaplan ◽  
GW Grant ◽  
JJ Toole ◽  
LL Leung
Keyword(s):  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Shear Rates ◽  
High Shear Rates ◽  
Dependent Inhibition ◽  
Platelet Thrombus

A novel thrombin inhibitor based on single-stranded (ss) deoxynucleotides with the sequence GGTTGGTGTGGTTGG (thrombin aptamer) has been recently discovered. In this study, we tested its efficacy in inhibiting clot-bound thrombin activity and platelet thrombus formation in an ex vivo whole artery angioplasty model. The thrombin aptamer showed a specific dose-dependent inhibition of thrombin-induced platelet aggregation (0.5 U/mL) in human platelet-rich plasma, with an IC50 of approximately 70 to 80 nmol/L. In an in vitro clot-bound thrombin assay system, heparin, used at clinically relevant concentrations of 0.2 U/mL and 0.4 U/mL, was ineffective in inhibiting clot-bound thrombin (6.5% and 34.9% inhibition at 0.2 U/mL and 0.4 U/mL, respectively). In contrast, the thrombin aptamer at an equivalent anticoagulant concentration inhibited clot-bound thrombin (79.7% inhibition). In an ex vivo whole artery angioplasty model, the thrombin aptamer markedly suppressed the generation of fibrinopeptide A (FPA), whereas heparin at 2 U/mL was ineffective. Compared with a scrambled ssDNA control, the thrombin aptamer reduced platelet deposition by 34.5% +/- 5% (mean +/- SEM, n = 4, P = .09) at low shear rates (approximately 200 s-1) and 61.3% +/- 11% (mean +/- SEM, n = 4, P = .05) at high shear rates (approximately 850 s-1). Thrombin aptamers based on ssDNA molecules represent a new class of thrombin inhibitors with potent anticoagulant and antithrombotic properties.

The contribution of thrombin-induced platelet activation to thrombus growth is diminished under pathological blood shear conditions

2012 ◽  
Vol 107 (02) ◽  
pp. 328-337 ◽  
Author(s):  
Hannah Lee ◽  
Sharelle Sturgeon ◽  
Shaun Jackson ◽  
Justin Hamilton
Keyword(s):  
Platelet Activation ◽  
Whole Blood ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Direct Result ◽  
Protease Activated Receptors ◽  
Heart Attacks ◽  
Shear Rates ◽  
Coronary Syndromes

SummaryDeveloping novel anti-platelet therapies is an important clinical strategy for the prevention of arterial thromboses which cause heart attacks and most strokes. Thrombin activates platelets via protease-activated receptors (PARs), and PAR antagonists are currently under investigation as antithrombotics. Yet despite these clinical advances, the importance of PARs to platelet activation during thromboses formed under pathological conditions has not been investigated. To this end, we examined the role of PAR-dependent platelet activation in thrombus formation in the presence of elevated blood shear rates. We used two in vivo thrombosis models and an ex vivo whole blood flow approach in PAR4-/-mice, whose platelets are unresponsive to thrombin, to show that the contribution of PAR-mediated platelet activation to thrombosis is diminished at pathological blood shear rates as a direct result of decreased incorporation of thrombin-activated platelets into growing thrombi. Our ex vivo observations were replicated in human whole blood treated with a PAR1 antagonist. These results define a novel, shear-regulated role for thrombin/PAR-dependent platelet activation during thrombosis and provide important insights into the conditions under which PAR antagonists may best be used for the prevention of acute coronary syndromes.

ANTI-THROMBOTIC EFFECTS OF E-5510 IN EXPERIMENTAL THROMBOSIS MODELS

1987 ◽  
Author(s):  
T Fujimori ◽  
T Saeki ◽  
K Harada ◽  
M Sato ◽  
N Ohshima
Keyword(s):  
Blood Flow ◽  
Oral Administration ◽  
Guinea Pigs ◽  
Vessel Wall ◽  
Pulmonary Thromboembolism ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Platelet Thrombus

A new agent developed in our laboratory, 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid (E-5510), suppressed various human platelet functions in vitro. The compound also showed quite potent ex vivo anti-platelet effects in many experimentalanimals. It is well known that anti-platelet effects are not always parallel to anti-thrombotic effects. Thus, in order to predict the efficacy of E-5510 in thrombotic disorders, the anti-thrombotic effects were examined in 3 different animal models of thrombosis.(1) Anti-thrombotic effect in an extracorporeal shunt model Two hrs after oral administration of the drug to guinea pigs,an extracorporeal shunt between the right carotid artery and the left jugular vein was performed. The thrombus formation on a silk thread inserted in the shunt tube was quantitated by measuring the time from the onset of circulation to the stenosis of blood flow. E-5510 dose-dependently inhibited thrombus formation, the minimum effective dose being 0.03 mg/kg.(2) Effect on microthrombus formation in mesenteric arteriole In order to evaluate the effect on intravascular platelet thrombus formation, thrombosis was induced in vivo in mesenteric arteriole in guinea pigs with filtered light from a mercury lamp and an intravenous fluorescent dye in an intravital microscope system (M. Sato and N. Ohshima, Thromb. Res.,35, 319, 1984). The thrombus formation was quantitated by measuring the time taken for circulating platelets to begin to adhere to vessel wall and the time taken for blood flow to stop completely due to fully developed thrombus. Both the time required for platelet adhesion to vessel wall and for platelet thrombus formation were significantly prolonged after oral administration of E-5510.(3) Effect on pulmonary thromboembolism Acute pulmonary thromboembolism was induced in mice by a bolus intravenous injection of arachidonic acid, and mortality was determined 3 min later. E-5510 dose-dependently reduced pulmonary thromboembolic mortality, and its ED50 was 0.11 mg/kg. The results described above indicate thatE-5510 may have beneficial effects in clinical treatments of thrombotic disease.

scholarly journals A novel nucleotide-based thrombin inhibitor inhibits clot-bound thrombin and reduces arterial platelet thrombus formation

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 677-682 ◽  
Author(s):  
WX Li ◽  
AV Kaplan ◽  
GW Grant ◽  
JJ Toole ◽  
LL Leung
Keyword(s):  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Shear Rates ◽  
High Shear Rates ◽  
Dependent Inhibition ◽  
Platelet Thrombus

Abstract A novel thrombin inhibitor based on single-stranded (ss) deoxynucleotides with the sequence GGTTGGTGTGGTTGG (thrombin aptamer) has been recently discovered. In this study, we tested its efficacy in inhibiting clot-bound thrombin activity and platelet thrombus formation in an ex vivo whole artery angioplasty model. The thrombin aptamer showed a specific dose-dependent inhibition of thrombin-induced platelet aggregation (0.5 U/mL) in human platelet-rich plasma, with an IC50 of approximately 70 to 80 nmol/L. In an in vitro clot-bound thrombin assay system, heparin, used at clinically relevant concentrations of 0.2 U/mL and 0.4 U/mL, was ineffective in inhibiting clot-bound thrombin (6.5% and 34.9% inhibition at 0.2 U/mL and 0.4 U/mL, respectively). In contrast, the thrombin aptamer at an equivalent anticoagulant concentration inhibited clot-bound thrombin (79.7% inhibition). In an ex vivo whole artery angioplasty model, the thrombin aptamer markedly suppressed the generation of fibrinopeptide A (FPA), whereas heparin at 2 U/mL was ineffective. Compared with a scrambled ssDNA control, the thrombin aptamer reduced platelet deposition by 34.5% +/- 5% (mean +/- SEM, n = 4, P = .09) at low shear rates (approximately 200 s-1) and 61.3% +/- 11% (mean +/- SEM, n = 4, P = .05) at high shear rates (approximately 850 s-1). Thrombin aptamers based on ssDNA molecules represent a new class of thrombin inhibitors with potent anticoagulant and antithrombotic properties.

scholarly journals Relative antithrombotic effects of monoclonal antibodies targeting different platelet glycoprotein-adhesive molecule interactions in nonhuman primates

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3218-3224 ◽  
Author(s):  
Y Cadroy ◽  
SR Hanson ◽  
AB Kelly ◽  
UM Marzec ◽  
BL Evatt ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Venous Flow ◽  
Shear Rates ◽  
Platelet Deposition ◽  
Antithrombotic Effects ◽  
Induced Aggregation

Abstract The relative antithrombotic effectiveness of targeting glycoprotein (GP) Ib-dependent versus GPIIb-IIIa-dependent platelet interactions has been determined in baboons by measuring thrombus formation after infusing comparable antihemostatic doses of anti-von Willebrand factor (vWF) monoclonal antibody (MoAb) BB3-BD5, anti-GPIb MoAb AP1, and anti- GPIIb-IIIa MoAb LJ-CP8 under conditions of arterial and venous flow (shear rates of 750 to 1,000 seconds-1 and 100 seconds-1, respectively). Thrombus formation was quantified as 111In-platelet deposition and 125I-fibrin accumulation on segments of collagen-coated tubing interposed in chronic exteriorized arteriovenous (AV) shunts for 40 minutes. In vitro, anti-vWF MoAb BB3 BD5 (IgG) and anti-GPIb MoAb AP1 [IgG or F(ab)2 fragments] inhibited ristocetin-induced platelet aggregation (IC50 50 nmol/L and 1 mumol/L, respectively), but neither of these MoAbs blocked platelet aggregation induced by adenosine diphosphate (ADP) (P > .5). Conversely, anti-GPIIb-IIIa MoAb LJ-CP8 inhibited platelet aggregation induced by ADP (IC50 1 mumol/L, but failed to block ristocetin-induced platelet aggregation (P > .5). In vivo, the intravenous infusion of anti-vWF MoAb BB3 BD5 or anti-GPIIb- IIIa MoAb LJ-CP8 into baboons at doses that abolished corresponding agonist-induced aggregation ex vivo (bolus injections of 0.5 mg/kg and 10 mg/kg, respectively) prolonged template bleeding times from baseline values of 4.0 +/- 0.3 minutes to > 27 +/- 4 minutes, and to > 26 +/- 4 minutes, respectively (P 3 .001 in both cases), without affecting the peripheral platelet count (P > .5). However, injection of anti-GPIb MoAb AP1 [10 mg/kg as IgG or 1 mg/kg as F(ab)2 fragments] produced immediate irreversible thrombocytopenia (< 40,000 platelets/microL). Anti-GPIIb-IIIa MoAb LJ-CP8 abolished platelet deposition and fibrin accumulation on collagen segments under both arterial and venous flow conditions (P < .01 in all cases), whereas MoAb BB3 BD5 produced minimal inhibition of platelet deposition and no decrease in fibrin accumulation at arterial shear rates and undetectable antithrombotic outcomes at low shear. Thus, inhibiting GPIIb-IIIa-dependent platelet recruitment abrogates both thrombus formation and platelet hemostatic function at both venous and arterial shear rates. By contrast, interfering with GPIb-vWF-dependent platelet interactions abolishes platelet hemostatic function without producing corresponding antithrombotic effects.

scholarly journals Relative antithrombotic effects of monoclonal antibodies targeting different platelet glycoprotein-adhesive molecule interactions in nonhuman primates

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3218-3224 ◽  
Author(s):  
Y Cadroy ◽  
SR Hanson ◽  
AB Kelly ◽  
UM Marzec ◽  
BL Evatt ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Venous Flow ◽  
Shear Rates ◽  
Platelet Deposition ◽  
Antithrombotic Effects ◽  
Induced Aggregation

The relative antithrombotic effectiveness of targeting glycoprotein (GP) Ib-dependent versus GPIIb-IIIa-dependent platelet interactions has been determined in baboons by measuring thrombus formation after infusing comparable antihemostatic doses of anti-von Willebrand factor (vWF) monoclonal antibody (MoAb) BB3-BD5, anti-GPIb MoAb AP1, and anti- GPIIb-IIIa MoAb LJ-CP8 under conditions of arterial and venous flow (shear rates of 750 to 1,000 seconds-1 and 100 seconds-1, respectively). Thrombus formation was quantified as 111In-platelet deposition and 125I-fibrin accumulation on segments of collagen-coated tubing interposed in chronic exteriorized arteriovenous (AV) shunts for 40 minutes. In vitro, anti-vWF MoAb BB3 BD5 (IgG) and anti-GPIb MoAb AP1 [IgG or F(ab)2 fragments] inhibited ristocetin-induced platelet aggregation (IC50 50 nmol/L and 1 mumol/L, respectively), but neither of these MoAbs blocked platelet aggregation induced by adenosine diphosphate (ADP) (P > .5). Conversely, anti-GPIIb-IIIa MoAb LJ-CP8 inhibited platelet aggregation induced by ADP (IC50 1 mumol/L, but failed to block ristocetin-induced platelet aggregation (P > .5). In vivo, the intravenous infusion of anti-vWF MoAb BB3 BD5 or anti-GPIIb- IIIa MoAb LJ-CP8 into baboons at doses that abolished corresponding agonist-induced aggregation ex vivo (bolus injections of 0.5 mg/kg and 10 mg/kg, respectively) prolonged template bleeding times from baseline values of 4.0 +/- 0.3 minutes to > 27 +/- 4 minutes, and to > 26 +/- 4 minutes, respectively (P 3 .001 in both cases), without affecting the peripheral platelet count (P > .5). However, injection of anti-GPIb MoAb AP1 [10 mg/kg as IgG or 1 mg/kg as F(ab)2 fragments] produced immediate irreversible thrombocytopenia (< 40,000 platelets/microL). Anti-GPIIb-IIIa MoAb LJ-CP8 abolished platelet deposition and fibrin accumulation on collagen segments under both arterial and venous flow conditions (P < .01 in all cases), whereas MoAb BB3 BD5 produced minimal inhibition of platelet deposition and no decrease in fibrin accumulation at arterial shear rates and undetectable antithrombotic outcomes at low shear. Thus, inhibiting GPIIb-IIIa-dependent platelet recruitment abrogates both thrombus formation and platelet hemostatic function at both venous and arterial shear rates. By contrast, interfering with GPIb-vWF-dependent platelet interactions abolishes platelet hemostatic function without producing corresponding antithrombotic effects.

Axial Dependence of Collagen–Induced Thrombus Formation in Flowing Non-Anticoagulated Human Blood

1995 ◽  
Vol 73 (01) ◽  
pp. 126-131 ◽  
Author(s):  
Helge E Roald ◽  
Kjell S Sakariassen
Keyword(s):  
Blood Flow ◽  
Platelet Adhesion ◽  
Potent Inhibitor ◽  
Thrombus Formation ◽  
Collagen Fibrils ◽  
Downstream Location ◽  
Perfusion Chamber ◽  
Platelet Concentration ◽  
Platelet Thrombus ◽  
Platelet Thrombi

SummaryPlatelet thrombus formation on collagen fibrils is most pronounced at the upstream end of the surface, and it gradually decreases along the axis in parallel with the direction of the blood flow. This phenomenon, known as axial dependent platelet thrombus formation, is explained by the balance of the platelet supply to the surface and the consumption of platelets by growing thrombi.In the present study we have affected this balance by (A) inhibiting the growth of platelet thrombi by aspirin (ASA) or clopidogrel, and thus increasing the platelet concentration at the surface, and by (B) utilising blood from cigarette smokers, who have enhanced thrombus formation immediately after smoking, and thus decreasing the platelet concentration at the surface. Thrombus formation in non-anticoagulated blood was triggered by collagen fibrils positioned in a parallel-plate perfusion chamber at a wall shear rate of 2600 s_1which is characteristic for moderately stenosed arteries. Morphometrical assessment of thrombus formation was performed at axial positions of 1 and 13 mm downstream to the blood flow inlet at the collagen surface.Platelet-collagen adhesion and thrombus volume in blood from nonsmokers were decreased at the downstream location by 39% (p ≤0.0001) and by 60% (p ≤0.0001), respectively. However, increasing the platelet concentration at the surface by partially inhibiting the thrombus growth by ASA or clopidogrel, reduced substantially the axial decrease in platelet adhesion and thrombus volume. The largest reduction was observed with clopidogrel which was also the strongest inhibitor of the thrombus growth at both axial positions investigated. The corresponding figures in blood from smokers with enhanced thrombus formation were 38% (p ≤0.0001) and 72% (p ≤0.001). Thus, enhanced upstream platelet consumption increased the axial reduction in thrombus volume, but not in platelet adhesion.These data substantiate the view that the “axial dependence phenomenon” may be explained by the balance between the platelet supply to the surface and the consumption of platelets by growing thrombi. It is also apparent that clopidogrel is a more potent inhibitor of platelet thrombus formation than ASA.

Studies on the Haemostatic Defect in a Complicated Syndrome

1995 ◽  
Vol 74 (05) ◽  
pp. 1244-1251 ◽  
Author(s):  
H Stormorken ◽  
H Holmsen ◽  
R Sund ◽  
K S Sakariassen ◽  
T Hovig ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Bleeding Tendency ◽  
Clot Retraction ◽  
Abnormal Finding ◽  
Shear Rates ◽  
Perfusion Chamber ◽  
Coagulant Activity ◽  
5 Ht Uptake ◽  
And Storage

SummaryThe Stormorken syndrome is a multifacetted syndrome including a bleeding tendency. No deviations were found in the coagulation- or fibrinolytic systems. Platelet number was low normal, and size abnormal, whereas EM findings were unremarkable. Survival time was half normal. Clot retraction was initially rapid, but clearly decreased, whereas prothrombin consumption was also initially rapid, but complete. Membrane GP’s were normal, so was AA metabolism, PI-cycle, granule storage and secretion, and c-AMP function, whereas 5-HT uptake and storage was decreased. Optical platelet aggregation was low normal with all physiological agonists. The only clearly abnormal finding was that coagulant activity was present on non stimulated platelets at the same level as kaolin-stimulated normal platelets. This indicated a platelet abnormality which should lead to a thrombogenic, not to a haemorrhagic trait. This paradox may have its origin in rheology, because when challenged with in vivo shear rates in an ex vivo perfusion chamber, platelet cohesion was abnormally low. Further studies to better delineate the membrane abnormality are underway.

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