Enhancing Effect of Collagen Receptor Polymorphisms on In Vitro Platelet Reactivity to Aspirin in Healthy Subjects.

Abstract [Background] Aspirin (ASA) is widely used as an antiplatelet drug, and a large number of clinical trials with ASA demonstrated significant efficacies for prevention and treatment of athrothrombosis. Recently, accumulating evidences indicated that there are inter-individual variations in the platelet response to ASA. The subpopulation, called ASA resistance, has the inability of response to ASA on ex vivo or in vitro platelet function tests and the poor clinical outcomes, although the mechanism underlying the variability is largely unknown. To date, genetic factors were showed to have an impact on platelet reactivity to ASA, and the inter-individual variations in platelet response to ASA was also reported to be associated with platelet sensitivity to collagen. In this study, the association between collagen-induced platelet aggregation (CIPA) and genetic polymorphisms of collagen receptors, glycoprotein (GP) Ia and GPVI, was analyzed using platelets treated by ASA (ASA +/−). We also investigated the effect of these polymorphisms on platelet thromboxane (TXB2) levels, closely related to the final stages of the arachidonate pathway inhibited by ASA. [Methods] We recruited genetically unrelated Japanese males (n=172) at their regular checkups. The mean age was 46.7±5.1 years. The subjects had no apparent hematologic or vascular disease and were not taking any medications that affect platelet function. Written informed consent was obtained from all study subjects. Platelet-rich plasma (PRP) sample was incubated with ASA [final concentration (fc) 10μM] or vehicle for 30 min at 24 degree Centigrade, and CIPA (fc 2μg/ml) test was performed on each PRP sample. Subsequently, platelet TXB2 levels were measured in the supernatant after centrifugation of each sample of CIPA test. Genotypes of the 807TC, Glu534Lys, Asn927Ser polymorphisms of GPIa and the Ser219Pro, Lys237Glu, Thr249Ala, Gln317Leu, His322Asn polymorphisms of GPVI were determined using the single-nucleotide primer extension-based method. [Results] To examine the sensitivity of platelets to ASA in vitro, we analyzed CIPA and platelet TXB2 levels in ASA(+/−). The maximum platelet aggregation and TXB2 levels in ASA(+) were significantly lower than those in ASA(−) (paired t-test, p<0.0001 and p<0.0001, respectively). Next, we investigated the association between the collagen receptor polymorphisms and the maximum platelet aggregation in ASA (+/−). For ASA(−), all genotypes of GPIa and GPVI were not associated with the maximum platelet aggregation. For ASA(+), subjects with 807TT/TC of GPIa had higher aggregation compared to those with 807CC(P=0.0135) whereas no association was observed between other polymorphisms and the maximum platelet aggregation. Moreover, repeated measures ANOVA showed that the difference in this inhibitory effect of ASA was significant between the 807TT/TC and 807CC genotypes (p=0.0253); the 807CC genotype has higher inhibitory effect of ASA. There was no association between platelet TXB2 levels and the GPIa and GPVI polymorphisms both in ASA(+) and ASA(−). [Conclusion] The 807CC genotype of GPIa polymorphism is associated with higher sensitivity to ASA in CIPA.

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Effects of Ticlopidine of Platelet Function in Men with Stable Angina Pectoris

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660138 ◽

1985 ◽

Vol 54 (04) ◽

pp. 808-812 ◽

Cited By ~ 7

Author(s):

Ulf Berglund ◽

Henning von Schenck ◽

Lars Wallentin

Keyword(s):

Platelet Aggregation ◽

Angina Pectoris ◽

Platelet Function ◽

Plasma Levels ◽

Platelet Reactivity ◽

Inhibitory Effect ◽

Controlled Study ◽

Double Blind ◽

Platelet Factor

SummaryThe effects of ticlopidine (T) (500 mg daily) on platelet function were investigated in a double-blind placebo-controlled study in 38 middle-aged men with stable incapacitating angina pectoris. The in vitro platelet reactivity to aggregating agents, the platelet sensitivity to prostacyclin and the plasma levels of platelet specific proteins and fibrinogen were determined before and after 4 and 8 weeks of treatment. T exerted a potent inhibitory effect on ADP- and collagen-induced platelet aggregation. The effect of T was proportional to the pretreatment reactivity to ADP and collagen. The inhibitory effect of T on the epinephrine response was less pronounced. The plasma levels of beta-thromboglobulin, platelet factor 4 and fibrinogen were not influenced by T. The platelet inhibition of prostacyclin was potentiated by T, and it was demonstrated that T and prostacyclin had synergistic inhibitory effects on platelet aggregation.

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A Comparison of the Effect of Decorsin and Two Disintegrins, Albolabrin and Eristostatin, on Platelet Function

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649933 ◽

1995 ◽

Vol 74 (05) ◽

pp. 1316-1322 ◽

Cited By ~ 13

Author(s):

Mary Ann McLane ◽

Jagadeesh Gabbeta ◽

A Koneti Rao ◽

Lucia Beviglia ◽

Robert A Lazarus ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Sequence Similarity ◽

Platelet Aggregation Inhibitors ◽

Antiplatelet Drug ◽

Rgd Sequence ◽

Fibrinogen Receptor ◽

Activated Platelets

SummaryNaturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP- induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin- activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the (β3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 μg/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

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The Effect of Barbituric Acid Derivatives on Platelet Function in Vitro and in Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649080 ◽

1973 ◽

Vol 30 (02) ◽

pp. 315-326

Author(s):

J. Heinz Joist ◽

Jean-Pierre Cazenave ◽

J. Fraser Mustard

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Barbituric Acid ◽

Platelet Rich Plasma ◽

Inhibitory Effect ◽

Primary Response ◽

Sodium Pentobarbital ◽

Barbituric Acid Derivatives

SummarySodium pentobarbital (SPB) and three other barbituric acid derivatives were found to inhibit platelet function in vitro. SPB had no effect on the primary response to ADP of platelets in platelet-rich plasma (PRP) or washed platelets but inhibited secondary aggregation induced by ADP in human PRP. The drug inhibited both phases of aggregation induced by epinephrine. SPB suppressed aggregation and the release reaction induced by collagen or low concentrations of thrombin, and platelet adherence to collagen-coated glass tubes. The inhibition by SPB of platelet aggregation was readily reversible and isotopically labeled SPB did not become firmly bound to platelets. No inhibitory effect on platelet aggregation induced by ADP, collagen, or thrombin could be detected in PRP obtained from rabbits after induction of SPB-anesthesia.

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Inhibition of Platelet Function by Antiarrhythmic Drugs, Verapamil and Disopyramide

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657151 ◽

1982 ◽

Vol 47 (02) ◽

pp. 150-153 ◽

Cited By ~ 13

Author(s):

P Han ◽

C Boatwright ◽

N G Ardlie

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Antiarrhythmic Drugs ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Second Phase ◽

Ionophore A23187 ◽

High Concentrations ◽

Artery Disease

SummaryVarious cardiovascular drugs such as nitrates and propranolol, used in the treatment of coronary artery disease have been shown to have an antiplatelet effect. We have studied the in vitro effects of two antiarrhythmic drugs, verapamil and disopyramide, and have shown their inhibitory effect on platelet function. Verapamil, a calcium channel blocker, inhibited the second phase of platelet aggregation induced by adenosine diphosphate (ADP) and inhibited aggregation induced by collagen. Disopyramide similarly inhibited the second phase of platelet aggregation caused by ADP and aggregation induced by collagen. Either drug in synergism with propranolol inhibited ADP or collagen-induced platelet aggregation. Disopyramide at high concentrations inhibited arachidonic add whereas verapamil was without effect. Verapamil, but not disopyramide, inhibited aggregation induced by the ionophore A23187.

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Effect Of Factor XIII On Platelet Aggregation. Study Of Platelet Function And Fibrin Stabilization Inhibitors

10.1055/s-0038-1653294 ◽

1981 ◽

Author(s):

M Maamer ◽

O Demay ◽

M Aurousseau

Keyword(s):

Platelet Aggregation ◽

Acetylsalicylic Acid ◽

Platelet Function ◽

Factor Xiii ◽

Platelet Rich Plasma ◽

Inhibitory Effect ◽

Photometric Method

There is little information on the participation of Factor XIII in platelet aggregation. Using BORN’s photometric method to study platelet aggregation induced by ADP in vitro on platelet rich plasma (PRP) of rabbit; clot solubility in 1 % monochloracetic acid and incorporation of dansylcadaverin into casein (LORAND L. et al.) to measure plasma FXIII concentration ; we showed that addition of activated F.XIII (F.XIIIa) to a PRP, aggregating power of platelets was significantly increased (+ 30.4 %, p<0.00l). Addition of inactive F.XIII or thrombin + Ca++ in concentrations used to activate F.XIII, had no significant effect on platelet aggregation induced by ADP.When F.XIIIa was added to plasma in presence of F.XIII inhibitors as 3178 AQ (a new synthetic benzothiophen keton derivative) or monodansylcadaverin (DC) in concentrations of (3.27 × 10-4 M and 9.31 × 10-4 m respectively), the platelet aggregation was significantly inhibited (- 48.8 % and - 35.4 % respectively, p<0.001). This inhibitory effect was not seen when dipyridamole or Acetylsalicylic Acid (ASA) in concentrations of (6.18 × 10-4 M and 17.3 × 10-4 M respectively) ware added in PRP in presence of F.XIIIa When platelet aggregation was performed without addition of F.XIIIa the inhibitory effect of 3178 AQ and DC was respectively (- 76.6 % and - 65.1 %, p<0.001), dipyridamole (- 37.6 %, p<0.00l) and ASA (-4.1%, no significant)These results suggest that F.XIIIa increased the platelet aggregation induced by ADP and compounds which are both inhibitors of platelet aggregation and F.XIII would be more potent antithrombotic by acting on platelets and fibrin stabilization, than drugs which are inhibitors of platelet aggregation only.

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Novel 12-LOX Inhibitor ML355 Attenuates Platelet Reactivity and Impairs Thrombus Growth, Stability and Vessel Occlusion In Vivo

Blood ◽

10.1182/blood.v126.23.3442.3442 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3442-3442 ◽

Cited By ~ 1

Author(s):

Reheman Adili ◽

Theodore R Holman ◽

Michael Holinstat

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Platelet Adhesion ◽

Ex Vivo ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Vessel Occlusion ◽

Growth Stability

Abstract Background: Adequate platelet reactivity is required for platelet adhesion and aggregation at the site of vascular injury to maintain hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi, the predominate underlying cause of myocardial infarction and stroke. While current anti-platelet treatments limit platelet function, they often result in an increased risk of bleeding. 12-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated by our lab and others to regulate PAR4 and GPVI-mediated platelet reactivity suggesting a role of 12-LOX in regulation of vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Aims: To determine how 12-LOX regulates thrombus formation in vivo and whether platelet 12-LOX is an effective target for anti-platelet therapeutics, wild-type (WT) or 12-LOX deficient (12-LOX-/-) mice were treated with or without the 12-LOX inhibitor, ML355, and were assessed for inhibitory effects on platelet activation in vitro, ex-vivo and in vivo. Methods: The effect of the novel 12-LOX inhibitor ML355 on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber. In vivo thrombus formation and vessel occlusion in small and large vessels were studied in 12-LOX-/-, WT mice and mice treated with ML355 using intravital microscopy using the FeCl3 injury models. Results: Using in vitro platelet aggregation assays, ML355 dose dependently inhibited thrombin, PAR1-AP, and PAR4-AP-induced aggregation in washed human platelets. Interestingly, the negative regulatory effects of ML355 inhibition of 12-LOX can be overcome by high concentration of thrombin. Additionally, ML355 was able to attenuate ADP-induced platelet aggregation both in platelet-rich-plasma and whole blood. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX-/- mice was impaired in FeCl3-induced mesenteric or carotid artery thrombosis models. Thrombi in 12-LOX-/- mice were unstable and frequently form emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The highly selective 12-LOX inhibitor ML355 inhibits platelets aggregation induced by various platelet agonists and ML355 inhibition of platelet function is not agonist specific. Platelet function at high shear in ex vivo conditions in both mice and human was attenuated in the presence of ML355. Thrombus growth, stability, and vessel occlusion was impaired in mice deficient for 12-LOX. Finally, the highly selective 12-LOX inhibitor ML355 attenuates thrombus formation and prevents vessel occlusion in vivo. Our data strongly indicates 12- LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics. Disclosures No relevant conflicts of interest to declare.

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Inhibition of platelet function by organic nitrate vasodilators

Blood ◽

10.1182/blood.v55.4.649.bloodjournal554649 ◽

1980 ◽

Vol 55 (4) ◽

pp. 649-654

Author(s):

AI Schafer ◽

RW Alexander ◽

RI Handin

Keyword(s):

Myocardial Ischemia ◽

Platelet Function ◽

Coronary Circulation ◽

Platelet Reactivity ◽

Inhibitory Effect ◽

Organic Nitrate ◽

Artery Disease ◽

In Vitro Effects ◽

External Calcium

There is evidence that platelet activation in the coronary circulation may be important in the pathogenesis of myocardial ischemia. Since organic nitrate vasodilators are commonly used in coronary artery disease, we have studied the in vitro effects of these drugs on platelet function. Nitroglycerin, isosorbide dinitrate, and their biotransformation product, inorganic nitrite, inhibited platelet aggregation with collagen, epinephrine, arachidonate, and ionophore, and blocked both primary and secondary aggregation in response to ADP. Nitroglycerin was studied in more detail. Its inhibitory effect was reversible and not dependent on external calcium concentration. It inhibited arachidonic acid oxygenation as measured by the arachidonate- induced oxygen burst and malonaldehyde production. These effects were not due to an increase in intracellular cyclic AMP. This unusual generalized inhibition of platelet function by nitroglycerin possibly contributes to its beneficial effect in myocardial ischemia in part by attenuating platelet reactivity in the coronary circulation.

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Possible Mode of Action of Ticlopidine: A Novel Inhibitor of Platelet Aggregation

10.1055/s-0039-1687403 ◽

1979 ◽

Cited By ~ 1

Author(s):

H. Johnson ◽

J. B. Heywood

Keyword(s):

Platelet Aggregation ◽

Mode Of Action ◽

Potent Inhibitor ◽

Platelet Reactivity ◽

Inhibitory Effect ◽

Thromboxane Synthetase ◽

Sustained Effect ◽

Dependent Inhibition

Ticlopidine (T) is weakly active in vitro, but is a potent inhibitor of platelet aggregation induced by ADP, collagen, thrombin, adrenaline, arachidonic acid, prostaglandin (PG) endoperoxide and thromboxane A2 with a sustained effect, when administered to a variety of animal species, including man. Platelets from treated animals were normal in ultrastructure and 14C-ADP binding was not modified by T. Basal PG synthesis was unaffected, whereas aspirin (A) had a marked inhibitory effect. Platelet cyclo-oxygenase and thromboxane synthetase activities were 90.6±12.9 and 96.1±5.3% of control following T treatment. In contrast to A, T had no effect on vascularprostacyclin (PGI2) synthesis, this being 1.4±0.1, 0.5±0.1 and 1.3±0. 3ng/mg wet weight aorta in T and A-treated and control animals respectively. Platelets from T-treated rats were significantly more responsive to inhibition by exogenous PGI2 (0.2-4 ng/ml) and PGE1 (4- 20 ng/ml). when compared with controls. T administration (30-300 mg/kg) resulted in a dose-dependent inhibition of ADP-induced platelet aggregation (26.0- 87. 5%) and enhancement of platelet reactivity to PGI2 (37.0-159.8%). There was a good correlation between these parameters (r=+0.994). T is a potent inhibitor of platelet aggregati on with a novel mode of action. It is not aspirin-like, but may act to potentiate endogenous PGI2 in vivo, possibly through an effect on platelet adenylate cyclase.

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PLATELET FUNCTION AND PLASMA FIBRINOGEN IN YOUNG SURVIVORS OF MYOCARDIAL INFARCTION

10.1055/s-0038-1643019 ◽

1987 ◽

Author(s):

U Berglund ◽

H von Schenk ◽

L Wallentin

Keyword(s):

Myocardial Infarction ◽

Platelet Function ◽

Platelet Reactivity ◽

Platelet Rich Plasma ◽

Inhibitory Effect ◽

Plasma Fibrinogen ◽

Women Patients ◽

Platelet Factor ◽

Young Survivors

An increased liability for thrombosis might be of pathogenetic importance in young survivors of myocardial infarction (MI). In 73 (58 men and 15 women) patients with MI below 45 years of age and 73 matched healthy controls plasma fibrinogen and platelet function tests were studied 3-6 months after the MI. At the time of the MI 77% of the patients were smokers but at the time of the investigation 27% of the patients smoked compared to 37% of the controls. Platelet aggregabi1ity was measured in vitro in platelet-rich plasma (PRP) as maximal aggregation to ADP and collagen. The platelet sensitivity to the inhibitory effect of prostacyclin (PG12) was tested by preincubation of PRP with PG12 before inducing aggregation with ADP 5 μM. Plasma levels of beta-thrombog1obuIin (BTG) and platelet factor 4 (PF4) were measured by RIA methods and plasma fibrinogen by heat precipitation. The table presents the results (means ± SE). * is p<0.04, ** is D<0.02 and ns is non significant.Severe emotional stress preceeding the MI occured in 7 patients - these cases had an increased platelet reactivity to ADP. The fibrinogen level was also elevated by smoking and obesity (multivariate analysis). Conclusion: young MI patients have elevated levels ol fibrinogen and reduced platelet sensitivity to PGI2. This might cause an increased thrombotic tendency.

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Individual Variation in Hemostatic Alterations Caused By Tyrosine Kinase Inhibitors - a Way to Improve Personalized Cancer Therapy?

Blood ◽

10.1182/blood.v128.22.1908.1908 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1908-1908

Author(s):

Kourosh Lotfi ◽

Suryyani Deb ◽

Clara Sjöström ◽

Anjana Tharmakulanathan ◽

Niklas Boknäs ◽

...

Keyword(s):

Platelet Aggregation ◽

Side Effects ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Platelet Function ◽

Thrombin Generation ◽

Kinase Inhibitors ◽

Thrombus Formation ◽

Individual Variations

Abstract Introduction During the last two decades, Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukemia (CML), and are now considered standard treatment for this disease. However, TKIs can induce serious hemostatic side effects including cardiovascular disease and bleeding disorders. Blood platelet aggregation and formation of pro-coagulant platelets are important to allow a well-balanced hemostatic response. Therefore, a detailed understanding of what effect different TKIs exert on platelets and hemostasis could help to understand if there are differences of importance to minimize the risk of bleeding complications in treated patients. Aim To investigate how TKIs used in CML (imatinib, dasatinib, nilotinib, bosutinib, and ponatinib) affect platelet activation and hemostasis. Materials and Methods We have developed a multi-parameter six color flow cytometry protocol to study different aspects of platelet function upon activation, e.g. formation of aggregatory (PAC-1-positive) and pro-coagulant (phosphatidylserine-exposing) platelets, exocytosis of alpha- and lysosomal granules and mitochondrial membrane potential.This protocol was performed in presence or absence of TKIs in blood from normal donors and in treated patients. Whole blood aggregometry (Multiplate®), thrombin generation in platelet-rich plasma and in vitro thrombus formation by free oscillation rheometry (ReoRox G2) was further evaluated in some situations. Results At clinically relevant concentrations, dasatinib significantly decreased the formation of procoagulant platelets. Ponatinib induced a slight decrease in formation of procoagulant platelets, whereas bosutinib and nilotinib showed opposite tendencies (n=7). Dasatinib also decreased platelet aggregation (n=4-6) and in vitro thrombus formation (n=3). Thrombin generation was not significantly affected by therapeutic levels of TKIs, whereas higher doses of dasatinib, bosutinib, ponatinib and imatinib significantly changed one or several of the thrombin generation parameters (n=7-8). Interestingly, large differences in response to the drugs were observed among the healthy donors, especially for dasatinib and bosutinib. Major inter-individual variations were also observed in dasatinib-treated patients. Conclusions Different TKIs show varying potency to affect platelet-based hemostasis. In addition, we found large inter-individual variations in how some drugs affected platelet function. Therefore, we suggest that development of a clinically useful protocol for platelet function testing could help to identify patients more susceptible to adverse drug reactions. Such a protocol could potentially help clinicians to gain insight into the risk of side effects, which could help to choose the most suitable drug for each individual patient. Disclosures No relevant conflicts of interest to declare.

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