| 1. |
- Nylander, Sven, et al.
(författare)
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Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP- and thrombin-induced human platelet activation
- 2004
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Ingår i: British Journal of Pharmacology. - : Wiley. - 0007-1188 .- 1476-5381. ; 142:8, s. 1325-1331
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Tidskriftsartikel (refereegranskat)abstract
- The objective of this study was to investigate if there is a synergistic effect of a combination of P2Y12 and P2Y1 inhibition and P2Y12 and thrombin inhibition, on ADP- and thrombin-induced platelet activation, respectively. The rationale being that these combinations will cause a concurrent inhibition of both Gαq and Gαi signalling.Blood from healthy volunteers was preincubated with AR-C69931MX, a reversible P2Y12 antagonist; MRS2179, a reversible P2Y1 antagonist; or melagatran, a direct reversible thrombin inhibitor; alone or in various combinations prior to activation with ADP or thrombin. Platelet function in whole blood was assessed by flow cytometry using the antibody PAC-1 to estimate the expression of active αIIbβ3 (the fibrinogen receptor GPIIb/IIIa). A synergistic effect was evaluated by comparing the concentrations in the different combinations with those of corresponding equipotent concentrations of each single inhibitor alone. The equipotent single concentrations were experimentally obtained from concentration response curves performed in parallel.A synergistic effect regarding inhibition of ADP-induced platelet activation (10 μM) was obtained with different combinations of AR-C69931MX and MRS2179.Inhibition of thrombin-induced platelet activation (2 nM) with combinations of AR-C69931MX and the thrombin inhibitor melagatran did also result in a strong synergistic effect.To our knowledge, this is the first time that data supporting a synergistic effect has been published for the inhibitor combinations described.Whether this synergistic effect in vitro also results in an improved antithrombotic effect in vivo with or without an increased risk of bleeding remains to be studied in well-conducted clinical studies.
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| 2. |
- Nylander, Sven, et al.
(författare)
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The relative importance of the ADP receptors, P2Y12 and P2Y1, in thrombin-induced platelet activation
- 2003
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Ingår i: Thrombosis Research. - : Elsevier BV. - 0049-3848 .- 1879-2472. ; 111:1-2, s. 65-73
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Tidskriftsartikel (refereegranskat)abstract
- The objective of this study was to test the relative importance of the two adenosine diphosphate (ADP) receptors P2Y1 and P2Y12 in thrombin-induced platelet activation using specific receptor antagonists. Blood from healthy volunteers was incubated with MRS2179, a reversible P2Y1 antagonist, or AR-C69931, a reversible P2Y12 antagonist prior to activation with different concentrations of ADP or thrombin. Platelet function in whole blood was assayed by flow cytometry using the antibody PAC-1 to estimate the expression of conformational active αIIbβ3, the fibrinogen receptor. Complete inhibition of P2Y12 or P2Y1 abolished the ADP response, but only inhibition of P2Y12 reduced the thrombin-induced response. The relative inhibition of the thrombin response by complete inhibition of P2Y12 was most pronounced at thrombin concentrations just enough for complete PAR1 cleavage, which is sufficient to release all ADP, giving 70–86% inhibition. Above this concentration, the relative importance of P2Y12 inhibition decreased due to activation of ADP independent pathways. This study supports P2Y12 as a drug target compared with P2Y1.
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| 3. |
- Ramström, Sofia, 1973-, et al.
(författare)
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Effects of inhibition of P2Y1 and P2Y12 on whole blood clotting, coagulum elasticity and fibrinolysis resistance studied with free oscillation rheometry
- 2003
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Ingår i: Thrombosis Research. - 0049-3848 .- 1879-2472. ; 109:5-6, s. 315-322
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: In vivo, initial platelet activation is likely caused by platelet contacts with collagen in the subendothelium or from the small amounts of thrombin formed by the tissue factor/factor VIIa complex. Our aim was to study the coagulative role of ADP released by the platelets after activation with strong stimuli such as collagen and/or thrombin, and the relative importance of the platelet ADP receptors P2Y1 and P2Y12.Materials and methods: We used 10 Hz free oscillation rheometry to measure clotting time, clot elasticity and fibrinolysis resistance of non-anticoagulated whole blood. The platelets were activated with a collagen-related peptide (CRP), with the PAR1 thrombin receptor activating peptide TRAP-6 or by thrombin, the latter generated by small amounts of thromboplastin. To inhibit the platelet ADP receptors, we used the P2Y1 antagonist MRS2179 and the P2Y12 antagonist AR-C69931MX.Results: Both antagonists significantly retarded the clotting induced by CRP. The effects were most pronounced with AR-C69931MX. For TRAP-6, the same trend was seen, but the retardation was only significant with AR-C69931MX. Clotting induced by small amounts of thromboplastin was not affected by any ADP-receptor antagonist. Addition of both antagonists did not change the results as compared to samples with AR-C69931MX alone. Nor did the antagonists, one at a time or in concert, effect fibrinolysis or the elastic properties of the clot.Conclusion: We conclude that ADP-receptor inhibition prolongs the clotting time for whole blood activated by CRP, but that it does not affect the properties of the subsequently formed coagulum.
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| 4. |
- Ramström, Sofia, 1973-, et al.
(författare)
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Platelet phosphatidylserine exposure and procoagulant activity in clotting whole blood : different effects of collagen,TRAP and calcium ionophore A23187
- 2003
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Ingår i: Thrombosis and Haemostasis. - 0340-6245 .- 2567-689X. ; 89:1, s. 132-141
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Tidskriftsartikel (refereegranskat)abstract
- We have studied the effects of different platelet agonists onphosphatidylserine (PS) exposure and clotting times in bloodwithout anticoagulants. Similar reductions in clotting time wereobtained for collagen, TRAP-6 or calcium ionophore A23187(50 µmol/L), in spite of huge differences in PS expression[6.7 ± 2.4%, 2.3 ± 0.5% and 99.9 ± 0.1%, respectively (mean ±SD, n = 5)]. Furthermore, the clotting times were much longerfor samples with A23187 exposing the same amounts of PS assamples with collagen or TRAP-6. Annexin V reversed theclotting time reduction, but could not prevent coagulation.Addition of phospholipid vesicles containing 20% PS neitheraffected the clotting times nor induced clotting in recalcified,platelet-free plasma.We conclude that platelet PS exposure is necessary, but notsufficient, for the coagulation amplification observed whenplatelets are stimulated via physiological receptors in a wholeblood environment.
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| 5. |
- Ramström, Sofia, 1973-, et al.
(författare)
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The role of platelets in blood coagulation : effects of platelet agonists and GPIIb/IIIa inhibitors studied by free oscillation rheometry
- 2002
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Ingår i: Thrombosis Research. - 0049-3848 .- 1879-2472. ; 105:2, s. 165-172
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Tidskriftsartikel (refereegranskat)abstract
- We have studied the contribution of platelets to the coagulation of plasma and the effects of activation or inhibition of platelets on the coagulation process in unanticoagulated fresh whole blood (subsequently termed native blood). For this purpose, we have used a free oscillation rheometer (FOR), the ReoRox4, a new instrument that enables noninvasive viscoelastic measurements of clot formation in plasma and whole blood. Platelets appear essential for the initiation of coagulation if no activating surface is present. We prepared platelet-free plasma by quick centrifugation and filtration of native blood, which did not coagulate if stored in plastic containers at 37 °C but clotted if transferred to glass containers. Addition of platelet agonists, such as collagen or the thrombin receptor agonist peptide, SFLLRN, significantly accelerated the clotting of native blood and also changed the rheometer curve appearance, accelerating both onset and completion of clot formation (i.e. fibrin gel formation). Inhibition of platelet glycoprotein (GP) IIb/IIIa with the peptide-derived compound MK-852 or the antibody-derived abciximab (Reopro) prevented clot retraction and prolonged the clotting time with SFLLRN. In collagen-stimulated samples, MK-852 accelerated clotting but delayed completion of clotting while abciximab prolonged both clotting time and completion of clotting. To our knowledge, this is the first report showing that activation of platelets in native whole blood shortens the coagulation time ex vivo. It also describes a new instrument that enables studies of the viscoelastic properties of a forming whole blood clot.
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| 6. |
- Ramström, Sofia, 1973-
(författare)
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The role of platelets in whole blood coagulation
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The body's first line of defence against bleeding is the platelets, small cell fragments with multiple roles in the coagulation process. The platelets are patrolling our blood vessels, looking for damage in the endothelial cell layer, to which they attach and transform, recruit more platelets and assemble the plasma coagulation proteases on their surfaces. Cleavage of the plasma coagulation factors then leads to the formation of a fibrin network sealing the damage.This thesis present studies of the role of platelets in whole blood coagulation, using methods which enable studies in a whole blood environment, flow cytometry and free oscillation rheometry (FOR). FOR proved to be equivalent in accuracy to visual inspection in detecting clotting, but allowing for simultaneous analysis of a large number of blood samples. A flow cytometry method for measuring the contents and release capacity of platelet dense granules was also developed.FOR was used to study platelets in coagulation. A quick removal of all blood cells was shown to be enough to keep the plasma anticoagulated. Activation of platelets by addition of collagen or a thrombin receptor agonist peptide (TRAP-6) shortened the whole blood clotting time, indicating the importance of platelets for the acceleration of coagulation. Inhibitors of the platelet fibrinogen receptor prevented clot retraction and prolonged the clotting time with TRAP-6. In collagen-stimulated samples, however, MK-852 accelerated clotting but delayed completion of clotting, while abciximab prolonged both clotting time and completion of clotting. Inhibitors to the platelet ADP receptors prolonged the clotting time, but did not affect the coagulum elasticity or the resistance against fibrinolysis. The same results were seen in a patient with a defective release of dense granule contents (which normally includes ADP). When comparing the platelet surface exposure of phosphatidylserine (PS) with the actual shortening of the clotting time, we found that despite the large decrease in clotting times seen with collagen or TRAP-6, only low numbers of PS-exposing platelets were detected. Induction of a similar PS exposure by the calcium ionophore A23187 could not decrease the clotting times to the same extent, indicating that PS exposure is only one of the mechanisms involved in platelet binding of plasma coagulation factors. Addition of annexin V, a substance that binds to PS, hampered, but could not abolish, coagulation. Artificial PS-containing lipid vesicles could not replace platelets, as they did not affect the coagulation of blood or plasma. To see which coagulation pathway that was involved in platelet dependent blood coagulation, inhibitors to factor XII or tissue factor (TF) were added to the blood prior to the platelet agonist Inhibition of factor xn increased the clotting time from about 10 to 15 minutes. Inhibition of TF was without effect. The results harmonised with the coagulation behaviour of blood congenitally deficient in factor XII or VII. Inhibition of factor XI caused a two to three fold increase in clotting time.We conclude that plasma does not necessarily coagulate if all blood cells are quickly removed. Activated platelets accelerate the coagulation of native whole blood ex vivo in a process which is independent of TF. Platelet PS, factor XI and factor XII all promote this coagulation, but are not essential This suggests that there might exist a platelet dependent blood coagulation pathway differing from the established pathways. Drugs affecting the platelet fibrinogen and ADP receptors affect the coagulation of the blood samples which might be detected by FOR. Our studies suggest the possibility of using FOR to measure the "platelet reactivity potential".
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| 7. |
- Rånby, Mats, et al.
(författare)
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Clotting time by free oscillation rheometry and visual inspection and a viscoelastic description of the clotting phenomenon
- 2003
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Ingår i: Scandinavian Journal of Clinical and Laboratory Investigation. - : Informa UK Limited. - 0036-5513 .- 1502-7686. ; 63:6, s. 397-406
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Tidskriftsartikel (refereegranskat)abstract
- An automated procedure for determination of clotting time in whole blood was validated by direct comparison with the reference method, visual clotting time determination. The procedure was based on a 10 Hz free oscillation rheometer (FOR) of our design, the ReoRox®4. Recalcified citrated blood samples (n=30), clotting in the range 4 to 20 min, were used in the validation. Every 30 s of the analysis, as the change in stiffness (ΔG*) of the sample was monitored by FOR, the sample cup was shortly removed from the FOR and its contents inspected for first signs of clotting, i.e. visual clotting time determination. Various FOR clotting criteria were attempted. Best correlation to visual clotting time was found when ΔG* reached 0.01 Pa, which yielded linear regression slope, intercept and r2 of 0.98, 0.09 min and 0.98, respectively. For comparison, six plasma samples were analyzed in the same way and gave almost the same results. The accuracy of the FOR determinations was checked by also analyzing, in parallel, portions of the sample with a conventional oscillation rheometer, a Bohlin VOR. The rationale is given for preferring ΔG* over G* as a FOR monitoring function in coagulation tests and for including median filtration of the primary FOR data. An extension of the FOR theory to include ΔG* and evidence in support of inhomogeneous blood clotting are also given.
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