Packham Ma

Platelet function assays.

The roles of platelets in hemostasis and arterial thrombosis involve their adherence to sites of vessel injury or ruptured atherosclerotic plaques, aggregation to form hemostatic plugs or thrombi, and acceleration of the coagulation cascade leading to the formation of thrombin. These roles of platelets are described in this review, hereditary platelet defects and other abnormalities associated with bleeding disorders are listed, and the various aggregating agents are discussed. A number of tests of platelet function are reviewed, including a description of their advantages and disadvantages: bleeding time determination; measurement of platelet aggregation in citrated platelet-rich plasma by recording changes in light transmission; measurement of platelet aggregation in citrated whole blood by impedance aggregometry; measurement of platelet-related hemostasis with the high shear Platelet Function Analyzer (PFA-100) system and the Ultegra Rapid Platelet Function Assay; use of the Cone and Plate(let) Analyzer to measure platelet adherence and aggregation under conditions of high shear; measurement of secretion of granule contents (ATP, 14C-serotonin, platelet factor 4, beta-thromboglobulin) and the formation of thromboxane B(2); and use of flow cytometry to assess the state of platelet activation (including conformational changes in membrane glycoproteins and surface expression of P-selectin and phosphatidylserine) ex vivo and in vitro following addition of agonists, and to measure levels of platelet membrane glycoproteins in the detection of inherited deficiencies.

Influence of Apyrase on Stability of Suspensions of Washed Rabbit Platelets

Summary The addition of apyrase to suspensions of washed rabbit platelets stored at 37° maintains their sensitivity to ADP-induced platelet aggregation. This appears to be due to the degradation of the ADP which accumulates; if not degraded, this ADP can make the platelets refractory to added ADP.

Platelets, Thrombosis and Drugs

SummaryThe development of thrombosis involves 4 main factors: the vessel wall, the formed elements of the blood, blood coagulation, and blood flow. In venous thrombosis the dominant mechanism is blood coagulation. In arterial thrombosis, however, the major part in both the initiation and growth of thrombi is played by the platelets.In selecting drugs which inhibit platelet function it is helpful to know which of the platelet reactions that contribute to thrombus formation can be inhibited by various agents. Platelets adhere to the damaged vessel wall, collagen being probably the most important constituent involved. They are then stimulated to release the contents of their storage granules. Release-inducing agents promote the discharge of adenosine diphosphate (ADP) which causes platelets in the vicinity to swell to a more spherical shape, extend pseudopods and adhere to each other. Platelet aggregation is reversible, and a number of drugs have been shown to be capable of inhibiting platelet function at various stages, both in vitro and in vivo. Adrenaline, noradrenaline, oestrogens and nicotine enhance aggregation. Drugs which inhibit platelet function include the non-steroidal anti-inflammatory drugs, the pyrimido-pyrimidines (e.g. dipyridamole), hydroxychloroquine, clofibrate, and dextran.In this review the effects of drugs which inhibit platelet function are outlined and the extent to which they can be used to influence the course of thromboembolic disease in man is discussed. It is suggested that combinations of anti-platelet drugs with anticoagulants could prove clinically useful.

Response of platelets to tissue injury

Abstract The response of platelets to tissue injury involves the interaction of platelets with surfaces and the adherence of platelets to each other. When platelets are exposed to surfaces (such as collagen) or to particulate stimuli (such as antigen-antibody complexes, or polystyrene particles, viruses, or bacteria in the presence of γ-globulin), the platelets adhere to them and release some of their constituents. Among the materials released are ADP, AMP, serotonin, and a factor which increases the permeability of vessels in the microcirculation. The ADP can cause platelet aggregation. Sulfinpyrazone and anti-inflammatory drugs, such as phenylbutazone and acetylsalicylic acid, diminish the interaction of platelets with surfaces or particulate matter, decrease the extent of the release of platelet constituents, and inhibit platelet aggregation. Transient platelet aggregates, which can be produced in the myocardial or renal circulation by infusion of ADP, cause tissue injury and organ dysfunction. It is possible that the kidney changes which characterize the generalized Shwartzman reaction could involve platelet aggregates induced by endotoxin. Rejection episodes in human kidney transplants are associated with the formation of platelet aggregates in the kidney and can be reversed by administration of anti-inflammatory drugs such as cortisone and phenylbutazone.

Rapid clearance of procoagulant platelet‐derived microparticles from the circulation of rabbits

reactivity against b2 glycoprotein I in high and low salt enables the detection of high avidity antibodies that better correlate with thrombosis than low-avidity anti-b2 glycoprotein I antibodies. The improvement predominantly concerns the specificity of the assay. The presence of low avidity anti-b2 glycoprotein I antibodies in patient plasmas apparently obscures the correlation of the anti-b2 glycoprotein I IgG antibodies with thrombosis. Because of the limitations mentioned in the paragraph above, additional studies with other patient populations are needed to confirm our findings.

Synergism between platelet aggregating agents: the role of the arachidonate pathway.

Abstract Aggregation of platelets by low concentrations of ADP is augmented by non-aggregating concentrations of collagen, thrombin, arachidonate or the divalent cation ionophore A23,187. Release-inducing agents act synergistically with ADP and with each other. Both collagen and thrombin cause aggregation by releasing ADP and by freeing platelet arachidonate to form prostaglandin endoperoxides which give rise to thromboxane A2. In these experiments the role of the arachidonate pathway in the synergism between pairs of aggregating and release-inducing agents was examined. Indomethacin was used to inhibit conversion of arachidonate to prostaglandin endoperoxides and thromboxane A2 and creatine phosphate/creatine phosphokinase (CP/CPK) was used in some experiments to convert released ADP to ATP. Synergism of collagen with ADP, arachidonate or thrombin was inhibited by indomethacin indicating that the arachidonate pathway plays a major role in the synergistic effects to which collagen contributes. Synergism of thrombin with collagen or arachidonate was inhibited by indomethacin but synergism of thrombin with ADP was only slightly affected. Indomethacin had little influence on the combined effects of these two agents on platelet aggregation. Thus it appears that the conversion of platelet arachidonate to prostaglandin endoperoxides and thromboxane A2 plays a minor part in the synergistic effects in which thrombin or A23,187 are involved. Thus, the non-steroidal anti-inflammatory drugs may have only limited use in inhibiting the contribution of thrombin and ADP to the formation of platelet thrombi at sites of vessel injury.

Procoagulant surface exposure and apoptosis in rabbit platelets: association with shortened survival and steady-state senescence

Summary.  Background: The signal(s) for removal of senescent platelets from the circulation are not fully understood; phosphatidylserine (PS) expression on platelets and another marker of apoptosis, loss of mitochondrial inner membrane potential (ΔΨm), have been implicated in platelet clearance. Objective: To investigate whether shortened platelet survival and steady‐state platelet senescence are associated with increased surface exposure of PS and ΔΨm collapse. Methods: Survival of in‐vitro biotinylated rabbit platelets treated with thrombin or Ca2+‐ionophore A23187 was tracked by flow cytometry after injection. Steady‐state platelet senescence was investigated by infusing biotin to label a platelet cohort. PS expression and ΔΨm of in‐vitro biotinylated platelets and of the aging platelet cohort biotinylated in‐vivo were measured by flow cytometry using annexin V‐FLUOS and the ΔΨm‐sensitive dye CMXRos, respectively. Results: Although PS expression, ΔΨm and survival of thrombin‐degranulated platelets were similar to those of control platelets, increasing concentrations of A23187 caused increased surface exposure of PS and progressive shortening of platelet survival; only one‐sixth of PS‐expressing platelets also exhibited ΔΨm loss. The cohort of senescent, biotinylated platelets remaining in the circulation at 96 h had increased exposure of PS and collapsed ΔΨm; of the 17% of PS‐expressing platelets, one‐third did not exhibit ΔΨm loss. There was also an increase in platelets with collapsed ΔΨm but not expressing PS. Conclusions: Platelets with shortened survival and senescent platelets have increased surface exposure of PS, that may be involved in their clearance. PS expression can occur independently of ΔΨm collapse and conversely, in aged platelets, ΔΨm loss can occur independently of PS expression.

Vessel injury, platelet adherence, and platelet survival.

T relationship among platelet survival, vessel injury, and thrombosis is of interest because of the role that platelets play in the development of atherosclerosis and its thromboembolic complications. Blood platelets do not adhere to normal endothelium. However, when a blood vessel is injured, platelets adhere to the injury site and, if blood flow is disturbed, thrombi can form on the injured area. In arteries, fresh thrombi are composed largely of aggregated platelets and are stabilized by fibrin, whereas in veins, although thrombi may be initiated by a mass of aggregated platelets in a valve pocket, the thrombi are mainly composed of red blood cells in a fibrin network. In large normal arteries that have not been previously injured in which blood flow is laminar, only a thin layer of platelets coats the subendothelium when the endothelium is lost. If flow is disturbed, for example at vessel orifices and branches, so that platelets are brought into close contact with each other, thrombus formation will occur because of the accumulation of aggregating agents released from the platelets and thrombin that is generated at the injury site. Thrombi are not static structures, but have been shown to undergo episodic formation and dissolution. Several processes contribute to the dissolution of thrombi. These include the force of flowing blood, platelet deaggregation, and lysis of fibrin. Theoretically, if injury to blood vessels is extensive or repeated, resulting in consumption of a significant proportion of the circulating platelets, a reduction in platelet survival would be expected. Thus, it is not surprising that vessel wall injury is a common feature of many conditions in which platelet survival is shortened (table 1). Although there are

Platelets, Thrombosis and Atherosclerosis

The interaction of platelets with the vessel wall can contribute to the early stages in the development of atherosclerosis through effects on smooth muscle cell proliferation, endothelial permeability, and possibly by causing vessel wall injury. Platelets are involved in the development of thrombi in response to vessel injury, and the repeated formation of platelet emboli and platelet-fibrin emboli from the mural thrombi may be one of the factors that cause clinical complications of atherosclerosis. Drugs which inhibit platelet function, particularly those that prolong shortened platelet survival (sulfinpyrazone and dipyridamole) may prove to be important in inhibiting the response of blood to vessel injury and thereby modifying the extent of atherosclerosis and its complications.

The effect of aspirin inhibition of PGI2 production on platelet adherence to normal and damaged rabbit aortae

Abstract These experiments were designed to investigate whether or not PGI2 production by vessel walls is responsible for their non-thrombogenic property. Adherence of rabbit platelets to the endothelium and sub-endothelium of the rabbit aorta was studied in vitro and in vivo when PGI2 production by the vessel wall was inhibited by aspirin. For in vitro studies, everted segments of rabbit aorta were mounted on a probe and rotated in a suspension of washed 51Cr-labeled platelets; the number of adherent platelets was calculated from the radioactivity associated with the platelets adherent to the segments. (This method measures the adherence of individual platelets, not platelet thrombi.) When adherence to the subendothelium was to be measured, the endothelium was removed by passage of a balloon catheter. The platelets and/or the vessel wall were pretreated with aspirin (1 mM and 2 mM respectively), washed and resuspended in fresh medium before adherence was measured. For in vivo studies, 51Cr-labeled platelets were injected into rabbits and the aortae were perfused and fixed in situ 20 min after administration of 25 or 100 mg of aspirin per kg. Platelet accumulation on both undamaged and de-endothelialized aortae was examined. PGI2-like activity produced by the vessel walls was measured by a bioassay; it was demonstrated that the aspirin treatment abolished the ability of the vessels to form PGI2, even when sodium arachidonate was supplied as a precursor. Both in vitro and in vivo prevention of PGI2 production did not affect the number of platelets that adhered to the endothelium or the subendothelium under these experimental conditions. Thus it is unlikely that the production of PGI2 by the vessel wall prevents platelets from adhering to the endothelium or subendothelium, although PGI2 may limit subsequent thrombus formation at an injury site.