Comparative analysis of hemostasis system state indicators in severe COVID-19

Introduction. Clinical experience in managing patients with a new coronavirus infection caused by the SARS-CoV-2 allowed to identify specific hemostasis disorders, and enables to introduce the concept of COVID-associated coagulopathy. The aim of the study was to assess the direction of coagulogram parameter changes, whole blood clotting parameters and characteristics of platelet and plasma hemostasis in patients with severe COVID-19. Materials and methods. The parameters of the hemostasis system were assessed using venous blood of 12 patients with severe COVID-19 and 16 healthy volunteers. The whole blood clotting process was investigated by low-frequency piezothromboelastography. The platelet count and indicators of spontaneous and ADP-induced platelet aggregation were estimated with the help of a laser platelet aggregation analyzer. Fibrinolytic activity of plasma, plasminogen activity, content of fibrinogen, D-dimer, PTT, APTT, PTI and INR were assessed. Results. An increased level of fibrinogen, a 6-fold increased D-dimer level, and increased PTT were found in patients with severe COVID-19. The patient platelets count was reduced by 51 % (p <0.05), spontaneous platelet aggregation remained at nearly normal level. Almost complete inhibition of ADP-induced platelet reactivity and inhibition of XIIa-dependent fibrinolysis was revealed, despite an increased by 19.3 % (p <0.05) plasminogen activity. Parameters of the whole blood coagulation process pointed a pronounced activation of platelet hemostasis, a significant intensification of the polymerization stage of clot formation and an increased intensity of clot lysis and retraction. Conclusion. The significant increase of D-dimer level and paradoxical inhibition of plasma fibrinolytic activity revealed by test of XIIa-dependent fibrinolysis (in contrast to the increased intensity of clot lysis when assessing the coagulation of whole blood) indicate the complex pathogenic mechanisms of coagulopathy caused by SARS-CoV-2 infection, and the involvement of blood cells and the vascular wall in the process of pathological thrombus formation.

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Effect of aspirin and sodium salicylate on thrombosis, fibrinolysis, prothrombin time, and platelet survival In rabbits with indwelling aortic catheters

Blood ◽

10.1182/blood.v61.2.353.353 ◽

1983 ◽

Vol 61 (2) ◽

pp. 353-361 ◽

Cited By ~ 20

Author(s):

M Cattaneo ◽

A Chahil ◽

D Somers ◽

RL Kinlough-Rathbone ◽

MA Packham ◽

...

Keyword(s):

Platelet Aggregation ◽

Prothrombin Time ◽

Whole Blood ◽

Fibrinolytic Activity ◽

Sodium Salicylate ◽

Thrombus Formation ◽

Significant Inhibition ◽

Weak Inhibitor ◽

Platelet Survival ◽

High Doses

Abstract We have studied the effect of different doses of aspirin on platelet function, PGI2 formation, platelet survival, thrombosis, fibrinolysis, and prothrombin time in rabbits with indwelling aortic catheters. The thrombi formed around indwelling aortic catheters were found to have a large fibrin component, and their formation was inhibited by heparin administration. Thus, in these experiments we examined the effect of aspirin (a weak inhibitor of thrombin-mediated platelet aggregation) under conditions in which thrombin was a major factor in the initiation and growth of the thrombi. Only very high doses of aspirin tended to inhibit thrombus formation over the 5-day period of observation, and a statistically significant inhibition of thrombus formation was produced by equivalent concentrations of sodium salicylate. The failure of high doses of aspirin to achieve a significant inhibition of thrombosis under the conditions of these experiments (whereas an equivalent dose of sodium salicylate was inhibitory) could be due to aspirin inhibition of PGI2 formation. Shortened platelet survival was not affected by aspirin treatment or the dose sodium salicylate that inhibited thrombus formation. The tendency to inhibit thrombus formation appeared to be unrelated to an effect on platelets but was associated with prolongation of the one-stage prothrombin time and increased whole blood fibrinolytic activity; doses of aspirin that inhibited platelet aggregation in response to sodium arachidonate or collagen, and PGI2 formation by the vessel wall, did not have a significant effect on the amount of thrombus present at 5 days. However, the high doses of aspirin that inhibited PGI2 formation were associated with a tendency to increased thrombus formation during the first 3 hr after insertion of the catheter. The results of these experiments show that when thrombin is an important factor in the formation of thrombi, aspirin is a weak inhibitor of thrombosis unless doses are used that provide sufficient salicylate to interfere with blood coagulation and promote whole blood fibrinolytic activity. These results also show that thrombus formation can be inhibited without an apparent change in platelet survival.

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Abstract 336: Metabolites Reflecting Fresh Venous Thrombus: Lactic Acid and Guanine Enhance Whole Blood Coagulation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.336 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Atsushi Yamashita ◽

Chihiro Sugita ◽

Sayaka Moriguchi-Goto ◽

Eriko Nakamura ◽

Kazunari Maekawa ◽

...

Keyword(s):

Lactic Acid ◽

Platelet Aggregation ◽

Blood Coagulation ◽

Whole Blood ◽

Thrombus Formation ◽

Venous Blood ◽

Blood Stasis ◽

Adenosine Monophosphate ◽

Venous Thrombus ◽

Flight Mass Spectrometry

Background: Thrombus formation is a multicellular dynamic process involving platelets, leukocytes, and erythrocytes. Recent studies demonstrated that microemvironment affects cellular metabolism and that metabolites can alter the cellular function. The present study aims to identify metabolites reflecting fresh venous thrombus and their role on thrombus formation in rabbit. Methods: We performed metabolomic analysis of rabbit venous blood and jugular venous thrombus 4 hours after endothelial denudation and blood stasis using capillary electrophoresis-time of flight mass spectrometry. Effects of the altered metabolites on blood coagulation and platelet aggregation were assessed with rotation thromboelastometry and platelet aggregometer. Results: The metabolomics analysis identified 226 metabolites (133 cationic and 93 anionic metabolites) in the venous blood and thrombus. The levels of 7 or 4 of them were significantly more (thrombus/ blood ratio >5) or less (thrombus/ blood ratio <1/2) in thrombus than those in blood, and the metabolites included glycolysis, nucleotides, and redox-related metabolites. Three metabolites were detectable only in the blood or venous thrombus. Among the metabolites (thrombus/ blood ratio >5), lactic acid and guanine dose-dependently enhanced whole blood clotting with thromboelastometry. Adenosine monophosphate inhibited collagen-induced platelet aggregation. Conclusion: The glycolysis, nucleotides, and redox-related metabolites may reflect fresh venous thrombus, and lactic acid and guanine may enhance blood coagulation in venous thrombus formation. The metabolic change could provide new insight into the process of venous thrombus formation.

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Platelet Aggregation on Extracellular Matrix: Effect of a Recombinant GPIb-Binding Fragment of von Willebrand Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649616 ◽

1993 ◽

Vol 70 (03) ◽

pp. 522-526 ◽

Cited By ~ 19

Author(s):

R Dardik ◽

Z M Ruggeri ◽

N Savion ◽

S Gitel ◽

U Martinowitz ◽

...

Keyword(s):

Extracellular Matrix ◽

Platelet Aggregation ◽

Von Willebrand Factor ◽

Whole Blood ◽

Platelet Reactivity ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Sem Analysis ◽

Von Willebrand ◽

Willebrand Factor

SummaryPlatelets in whole blood incubated on extracellular matrix (ECM) produced by bovine corneal endothelial cells under oscillatory flow conditions demonstrate extensive aggregate formation. Since both platelet-subendothelium and platelet-platelet interactions are mediated by von Willebrand factor (vWF), we used this system to examine the effect of a recombinant GPIb-binding fragment of vWF (designated RG12986), comprising residues 445-733 of the native vWF subunit, on platelet reactivity with ECM. The seven cysteines present in the RG12986 fragment were reduced and alkylated in order to achieve a monomeric conformation. The recombinant vWF fragment binds to unstimulated platelets in the absence of exogenous modulators. When added to platelet-rich plasma, it inhibits ristocetin-induced platelet agglutination. Binding of 51Cr-labeled platelets in reconstituted whole blood to ECM was inhibited by RG12986 in a dose dependent and saturable manner, with IC50 of 4 μM and maximal inhibition (about 70%) at 6 μM. Scanning electron microscope (SEM) analysis showed that addition of RG12986 to whole blood significantly inhibited platelet aggregation on ECM. The extent of inhibition observed with RG12986 at a final concentration of 4 μM was similar to that obtained with the cell adhesion peptide RGDS at the concentration of 0.1 mM. The ability of the RG12986 fragment to inhibit platelet aggregation on ECM is in agreement with the concept that blockade of vWF-GPIb interaction may inhibit further events leading to activation of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex and subsequent thrombus formation.

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Effect of aspirin and sodium salicylate on thrombosis, fibrinolysis, prothrombin time, and platelet survival In rabbits with indwelling aortic catheters

Blood ◽

10.1182/blood.v61.2.353.bloodjournal612353 ◽

1983 ◽

Vol 61 (2) ◽

pp. 353-361

Author(s):

M Cattaneo ◽

A Chahil ◽

D Somers ◽

RL Kinlough-Rathbone ◽

MA Packham ◽

...

Keyword(s):

Platelet Aggregation ◽

Prothrombin Time ◽

Whole Blood ◽

Fibrinolytic Activity ◽

Sodium Salicylate ◽

Thrombus Formation ◽

Significant Inhibition ◽

Weak Inhibitor ◽

Platelet Survival ◽

High Doses

We have studied the effect of different doses of aspirin on platelet function, PGI2 formation, platelet survival, thrombosis, fibrinolysis, and prothrombin time in rabbits with indwelling aortic catheters. The thrombi formed around indwelling aortic catheters were found to have a large fibrin component, and their formation was inhibited by heparin administration. Thus, in these experiments we examined the effect of aspirin (a weak inhibitor of thrombin-mediated platelet aggregation) under conditions in which thrombin was a major factor in the initiation and growth of the thrombi. Only very high doses of aspirin tended to inhibit thrombus formation over the 5-day period of observation, and a statistically significant inhibition of thrombus formation was produced by equivalent concentrations of sodium salicylate. The failure of high doses of aspirin to achieve a significant inhibition of thrombosis under the conditions of these experiments (whereas an equivalent dose of sodium salicylate was inhibitory) could be due to aspirin inhibition of PGI2 formation. Shortened platelet survival was not affected by aspirin treatment or the dose sodium salicylate that inhibited thrombus formation. The tendency to inhibit thrombus formation appeared to be unrelated to an effect on platelets but was associated with prolongation of the one-stage prothrombin time and increased whole blood fibrinolytic activity; doses of aspirin that inhibited platelet aggregation in response to sodium arachidonate or collagen, and PGI2 formation by the vessel wall, did not have a significant effect on the amount of thrombus present at 5 days. However, the high doses of aspirin that inhibited PGI2 formation were associated with a tendency to increased thrombus formation during the first 3 hr after insertion of the catheter. The results of these experiments show that when thrombin is an important factor in the formation of thrombi, aspirin is a weak inhibitor of thrombosis unless doses are used that provide sufficient salicylate to interfere with blood coagulation and promote whole blood fibrinolytic activity. These results also show that thrombus formation can be inhibited without an apparent change in platelet survival.

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Sex Differences in the Determinants of Fibrinolytic Activity

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614950 ◽

1998 ◽

Vol 79 (03) ◽

pp. 587-590 ◽

Cited By ~ 12

Author(s):

J. A. Cooper ◽

D. J. Howarth ◽

T. W. Meade ◽

G. J. Miller ◽

P. K. MacCallum

Keyword(s):

Plasminogen Activator ◽

Whole Blood ◽

Fibrinolytic Activity ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Clot Lysis ◽

Significant Difference ◽

Main Determinants ◽

Activator Inhibitor Type ◽

Pai 1

SummaryImpaired whole blood fibrinolytic activity (FA), measured by the dilute clot lysis time (DCLT), is associated with first episodes of ischaemic heart disease (IHD) in the Northwick Park Heart Study in men, especially under 55 years, and in women. In a community-based study to investigate possible determinants of the DCLT, and therefore to assess which fibrinolytic components might be predictors of first IHD events, we measured fibrinolytic variables in a sub-sample of 150 healthy adults (73 males, 77 females) randomly selected from a single general practice.Most of the variance in DCLT (68% in men, 63% in women) was explained by tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) activities. In multiple regression analysis there was a significant difference in the strength of the association of t-PA activity with DCLT in men compared to women (test for interaction p = 0.05), the association of t-PA activity with DCLT being significant in males but not in females. Plasma PAI-1 activity was strongly associated with DCLT in both sexes. There was no independent association of DCLT with plasma fibrinogen, t-PA antigen, other fibrinolytic inhibitors, body mass index, serum lipids or C-reactive protein.Plasma PAI-1 activity in females and both t-PA and PAI-1 activities in males are the main determinants of whole blood FA measured by DCLT. It is therefore likely that these modulators of the plasma fibrinolytic system are associated with the onset of first clinical episodes of IHD. Elevated levels of t-PA antigen were positively associated with DCLT after adjustment for age and sex and therefore indicate impaired rather than enhanced FA. Further studies of the association of FA with risk of IHD should include not only “global” measures but also assessment of t-PA and PAI-1 activities, particularly as our results suggest that their associations with IHD may differ in men and women.

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Effects of exercise and conditioning on clotting and fibrinolytic activity in men

Journal of Applied Physiology ◽

10.1152/jappl.1987.62.4.1416 ◽

1987 ◽

Vol 62 (4) ◽

pp. 1416-1421 ◽

Cited By ~ 49

Author(s):

E. W. Ferguson ◽

L. L. Bernier ◽

G. R. Banta ◽

J. Yu-Yahiro ◽

E. B. Schoomaker

Keyword(s):

Fibrinolytic Activity ◽

Blood Clotting ◽

Degradation Products ◽

Clot Lysis ◽

Plasma Clot ◽

Fitness Program ◽

Fibrin Degradation Products ◽

Before And After ◽

Plasmin Inhibitor ◽

Sedentary Subjects

Sixty healthy men in three physical fitness categories (sedentary, on no organized fitness program; joggers, running 5–15 miles/wk; and marathoners, running greater than 50 miles/wk) were evaluated for changes in blood clotting and fibrinolytic activity before and after maximum exercise on a treadmill according to the Bruce protocol. The rate of blood clotting, as measured by prothrombin times, partial thromboplastin times and thrombin times, was accelerated by exercise (all P less than 0.005). The ability of euglobulin clots and plasma clots to lyse incorporated 125I-fibrin, termed 125I-euglobulin clot lysis (IEL) and 125I-plasma clot lysis (IPCL), were used as indexes of fibrinolytic activity. Marathoners had greater increases in fibrinolytic activity with exercise (76% compared with 63% for joggers and 55% for sedentary subjects by IEL; 427% compared with 418% for joggers and 309% for sedentary subjects by IPCL; all P less than 0.05). Fibrin degradation products increased with exercise (P less than 0.005 for the total group of 60 subjects). The absolute concentrations of alpha 2-plasmin inhibitor, alpha 2-macroglobulin, and antithrombin III increased with exercise (all P less than 0.005), but when concentrations were corrected for acute shifts of plasma water during exercise, the quantity of these inhibitors actually decreased (all P less than 0.005). The changes in clotting assays with exercise were not significantly correlated with changes in whole blood lactate, blood pyruvate, or rectal temperatures. Fibrinolytic assays before and after exercise correlated poorly to moderately with blood lactates (IEL: r = 0.441 and r = 0.425, respectively; IPCL: r = 0.294 and r = 0.544, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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THE ANTITHROMBOTIC EFFECT OF PALM OIL IS CORRELATED WITH ITS CONTENTS OF VITAMIN E

10.1055/s-0038-1643804 ◽

1987 ◽

Author(s):

E G Hornstra ◽

A H Hennissen ◽

R Kalafusz ◽

D T S Tan

Keyword(s):

Fatty Acid ◽

Platelet Aggregation ◽

Palm Oil ◽

Whole Blood ◽

Arterial Thrombosis ◽

Edible Oils ◽

Thrombus Formation ◽

Saturated Fatty Acids ◽

Atp Release ◽

Significant Negative Correlation

Dietary saturated fatty acids are known to increase platelet aggregation and arterial thrombogenesis.We recently demonstrated, however, that palm oil, rich in saturated palmitic acid, has a distinct antithrombotic affect, which is associated with a decrease of the thromboxane-prostacyclin ratio in activated whole blood. To identify the antithrombotic component(s) of palm oil, seven palm oil fractions were prepared with comparable fatty acid compositions of the triglycerides but containing Various amounts of non-triglyceride material with different compositions.These fractions were fed to rats in amounts of 50 energy% for a period of 8 weeks, after which arterial thrombosis tendency was measured upon insertion of an aortic prosthesis, the aorta-loop. During loop insertion, 1 ml blood was collected in citrate for measuring platelet aggregation and ATP release in response to collagen, using the Chronolog whole blood lumi-aggregometer. Arterial thrombosis tendency was found to be negatively related to the total amount of non-triglyceride material in the various fractions (r = 0.78; p <0.05).No significant relationship was observed between arterial thrombus formation and the various sterols present in the non-triglyceride material.A significant negative correlation was found, however, with a-tocopherol (r = 0.86; p <.02). Collagen-induced platelet aggregation and ATP release in whole blood were not correlated to total amounts or α-tocopherol content of the non-triglyceride material.However, significant positive relationships were found between these platelet functions and the amountsof the various sterols (Campesterol: r = 0.70; P < 0.10 β-sitostero1 : r = 0.69; P <0.10. Cholesterol : r = 0.81; P < 0.05).These findings demonstrate that effects of edible oils on platelet function and arterial thrombogenesisare not only mediated by the fatty acid compostion of the triglycerides, but can also be determined by 'minor components', present in the non-triglyceride part of the oils.

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Biomechanical thrombosis: the dark side of force and dawn of mechano-medicine

Stroke and Vascular Neurology ◽

10.1136/svn-2019-000302 ◽

2019 ◽

Vol 5 (2) ◽

pp. 185-197 ◽

Cited By ~ 1

Author(s):

Yunfeng Chen ◽

Lining Arnold Ju

Keyword(s):

Platelet Aggregation ◽

Blood Clotting ◽

Thrombus Formation ◽

Dark Side ◽

Atherosclerotic Plaques ◽

Excessive Bleeding ◽

Glycoprotein Vi ◽

Device Implantation ◽

Platelet Binding ◽

Platelet Thrombus

Arterial thrombosis is in part contributed by excessive platelet aggregation, which can lead to blood clotting and subsequent heart attack and stroke. Platelets are sensitive to the haemodynamic environment. Rapid haemodynamcis and disturbed blood flow, which occur in vessels with growing thrombi and atherosclerotic plaques or is caused by medical device implantation and intervention, promotes platelet aggregation and thrombus formation. In such situations, conventional antiplatelet drugs often have suboptimal efficacy and a serious side effect of excessive bleeding. Investigating the mechanisms of platelet biomechanical activation provides insights distinct from the classic views of agonist-stimulated platelet thrombus formation. In this work, we review the recent discoveries underlying haemodynamic force-reinforced platelet binding and mechanosensing primarily mediated by three platelet receptors: glycoprotein Ib (GPIb), glycoprotein IIb/IIIa (GPIIb/IIIa) and glycoprotein VI (GPVI), and their implications for development of antithrombotic ‘mechano-medicine’ .

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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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Blockade of adenosine diphosphate receptors P2Y12 and P2Y1 is required to inhibit platelet aggregation in whole blood under flow

Blood ◽

10.1182/blood.v98.12.3340 ◽

2001 ◽

Vol 98 (12) ◽

pp. 3340-3345 ◽

Cited By ~ 112

Author(s):

Nancy A. Turner ◽

Joel L. Moake ◽

Larry V. McIntire

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Thrombus Formation ◽

Direct Shear ◽

Flow Conditions ◽

Initial Adhesion ◽

Platelet Deposition ◽

Adp Receptors ◽

Induced Aggregation ◽

Control Samples

Abstract Using heparinized whole blood and flow conditions, it was shown that adenosine 5′-diphosphate (ADP) receptors P2Y12 and P2Y1 are both important in direct shear-induced platelet aggregation and platelet aggregation subsequent to initial adhesion onto von Willebrand factor (vWf)–collagen. In the viscometer, whole blood was subjected to shear rates of 750, 1500, and 3000 s−1 for 30 seconds at room temperature. The extent of aggregation was determined by flow cytometry. The P2Y12antagonist AR-C69 931MX (ARMX) reduced shear-induced aggregation at these rates by 56%, 54%, and 16%, respectively, compared to control samples. Adenosine 3′,5′-diphosphate (A3P5P; P2Y1antagonist) inhibited shear-induced aggregation by 40%, 30% and 29%, respectively, compared to control samples. Blockade of both ADP receptors at 3000 s−1 with ARMX plus A3P5P further reduced the platelet aggregation by 41% compared to the addition of ARMX alone (57% compared to control samples). Using a parallel-plate flow chamber, whole blood was perfused over bovine collagen type 1 at a wall shear rate of 3000 s−1 for 60 seconds. Platelet deposition was quantified with epifluorescence video microscopy and digital image processing. Blockade of P2Y12 alone or blockade of P2Y1 alone did not reduce thrombus formation on vWf–collagen. In contrast, blockade of both P2Y12 and P2Y1 reduced platelet deposition by 72%. These results indicate that combinations of antagonists of the ADP receptors P2Y12 and P2Y1 are effective inhibitors of direct shear-induced platelet aggregation and of platelet aggregation subsequent to initial adhesion under flow conditions. Inhibitors of these pathways are potentially useful as antiarterial thrombotic agents.

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