Protocol for monitoring platelet inhibition

[0001] The invention relates to a protocol for monitoring the effectiveness of antiplatelet agents.

Background of the invention [0002] Blood is the body's circulating tissue that carries oxygen and nutrients to the tissues and removes carbon dioxide and various metabolic products for excretion. Whole blood consists of pale yellow or gray-yellow fluid, plasma in which red blood cells, white blood cells and platelets are suspended.

[0003] Accurate measurement of hemostasis, i.e. the patient's blood ability to coagulate and solubilize quickly and efficiently, is crucial for some surgical and medical procedures. Accelerated (rapid) and accurate detection of abnormal hemostasis is also particularly important in relation to appropriate treatment that should be given to patients suffering from hemostatic disorders and such which may require administration of anticoagulants, antifibrinolytic agents, thrombolytic agents, antiplatelet agents or blood components in an amount. which must be clearly identified after taking into account anomalies in terms of ingredients, cells or "factors" in the patient's blood that may contribute to a haemostatic disorder^froms.

[0004] Hemostasis is a dynamic, extremely complex process involving many interacting factors that include coagulation and fibrinolytic proteins, inhibitors, activators and cellular elements such as platelet cytoskeleton, platelet cytoplasmic granules and platelet cell surfaces. As a result, during activation, no factor remains static or works in isolation. Thus, to be complete, it is necessary to continuously measure all stages of the patient's haemostasis as a net product of whole blood components, in an uninsulated or static manner. To give an example of the consequences of measuring the isolated portion of hemostasis, let's assume that the patient has developed fibrinolysis, which was caused by the activation of plasminogen - a change into plasmin, an enzyme that breaks down the clot. In this case, a byproduct of this process is the fibrinogen degradation product (FDP), which behaves like an anticoagulant. If the patient is tested only for anticoagulation and is treated accordingly, the patient may remain at risk due to non-antifibrinolytic treatment.

[0005] The final result of the hemostasis process is the three-dimensional network of polymerized fibrin (fibrinogen) fibers, which together with the binding glycoprotein IIb / IIIa platelet receptor (GPIIb / IIIa) forms the final clot (FIG. 1). A unique feature of this network structure is that behaves like a strong elastic solid, resistant to deforming shear stress of circulating blood. The resistance of the final curd to the deforming shear stress is determined by the structure and density of the fibrin fiber network and the forces exerted by the participating plates.

[0006] Platelets have been shown to affect the mechanical strength of fibrin in at least two ways. First, by acting as a node of branching points, which significantly increases the hardness of the fibrin structure. Secondly, by applying a "pull" force on the fibers due to the contractility of platelet actomyosin, a muscle protein that is part of the cytoskeleton-mediated contractility apparatus. The strength of this contractility further increases the strength of the fibrin structure. The GPIIb / IIIa platelet receptor appears to be key in anchoring polymerized fibers to the base cytoskeletal shrink apparatus in activated plates, thereby mediating mechanical transfer.

[0007] In this way, a clot that develops and adheres to the damaged vascular system as a result of activated hemostasis, and resists the deforming shear stress of circulating blood, is an indispensable mechanical molding agent to provide a "temporary plug" that is resistant to shear stress blood circulating during healing of the vessel. The kinetics, strength and stability of the clot, which are its physical properties, to resist the deforming shear stress of circulating blood, determine its ability to perform work with hemostasis, which is to stop hemorrhage without allowing inappropriate thrombosis. This is exactly the measurement for which the Thrombelastograph® system (TEG®) described below was designed, which is the time it takes to pre-form fibrin, the time it takes the clot to reach maximum strength, the actual maximum strength and stability of the clot.

[0008] Hemostasis analyzer tools have been known since Professor Helmut Hartert developed such a device in Germany in 1940. One type of hemostasis analyzer is described in Common US Patent No. 5,223,227. WO0196879 describes methods for determining antiplatelet therapy using two blood samples. This tool, the TEG® haemostasis analyzer, monitors the elastic properties of blood after inducing clotting in a low shear environment reminiscent of poor venous blood flow. Patterns of changes in shear elasticity of the developing clot allow the determination of clot formation kinetics as well as strength and stability of the formed clot; in short, the mechanical properties of the developing clot. As described above, the kinetics, strength and stability of the clot provide information on the ability of the clot to perform "mechanical work," i.e. resisting deforming shear stress of circulating blood; essentially, the clot is an elementary hemostasis apparatus, and the TEG® analyzer measures the ability of the clot to perform mechanical work during its structural development. The TEG® system continuously measures all stages of patient haemostasis as a net product of whole blood components, in an uninsulated or static way from the moment of initiation of the test to the initial formation of fibrin, by the rate of clot strengthening and ultimately the clot strength by platelet fibrin binding by means of GPIIb / platelet receptors IIIa and clot lysis.

[0009] Platelets play a key role in mediating ischemic complications (thrombosis) in patients. The use of GPIIb / IIIa inhibitors in patients with thrombosis or in addition to percutaneous coronary angioplasty (PTCA) is rapidly becoming the standard of care. Inhibition of the GPIIb / IIIa receptor is a very powerful form of antiplatelet therapy that can reduce the risk of death and myocardial infarction, but can also cause a dramatic risk of hemorrhage. The reason for potential bleeding or failing to achieve an adequate level of therapeutic platelet inhibition is the algorithm adapted to the weight of the platelet blocker treatment that is being used, although there is considerable variation for individuals. This is a problem, due in part to differences in platelet count and variability in GPIIb / IIIa receptors per platelet and their ligand binding functions.

[0010] Since the clinical introduction of the chimeric mouse / human Fab c7E3 antibody fragment (abciximab, ReoPro®), several synthetic forms of GPIIb / IIIa antagonists such as Aggrastat® (Tirofiban) and INTEGRILIN® (eptifibatide) have also been approved resulting in more widespread and increased using GPIIb / IIIa inhibitor therapy in interventional cardiology procedures.

[0011] Prior to the introduction of the method and apparatus described in the above-mentioned US Patent Application No. 09/591, 371, there was no rapid, reliable, quantitative point-of-care test available for monitoring therapeutic platelet blocking. Although the turbidimetric aggregation assay was used to measure the level of platelet GPIIb / IIIa receptor blockade in small clinical and dose studies, its routine use in clinical use to determine the dose of GPIIb / IIIa receptor antagonists in individual patients was not feasible. Aggregation is time-consuming (more than an hour), expensive to perform, requiring specialized staff to perform it, and not readily available around the clock; therefore, it cannot be used at care points for routine patient monitoring and dose individualization. To be clinically useful, a platelet inhibition test must provide fast and reliable information about receptor blockade at the patient's bed, thus enabling dose modification to achieve the desired antiplatelet effect.

[0012] Turbidimetric aggregation test, based on a photometric rule, monitors changes in optical density of the sample. Initially, a minimum amount of light passes through the sample, and functional platelets are activated by a turbidimetric test; platelet aggregation occurs via the platelet receptor GPIIb / IIIa and fibrin (fibrinogen) binds as shown in Figure 1, and thus light transmission increases. When platelets are inhibited by blocking the GPIIb / IIIa receptor, light transmission increases proportionally.

[0013] Another commercially available system measures fibrinogen-platelet binding using beads coated with a fixed amount of external "normal" fibrinogen source. Therefore, this system uses a source of "normal" non-human fibrinogen and is unable to detect the patient in a thrombotic state (hypercoagulability) due to a higher level of fibrinogen in the patient, or to detect a hemorrhagic condition (low blood coagulability) due to on a patient's low fibrinogen level. In addition, this system only shows the binding without detecting the breaking of this binding. Therefore, in the presence of thrombolysis, the assessment of platelet GPIIb / IIIa receptor blocking by the system cannot be accurate.

[0014] Fibrinogen-platelet binding GPIIb / IIIa is the initial stage of platelet aggregation, or the primary hemostatic platelet plug, which is transformed to form the final fibrin-platelet binding. Thus, measuring only the initial stage of fibrinogen-platelet binding is not sufficient, as it may not reflect the final binding, via GPIIb / IIIa receptor, of fibrinogen platelet. Because turbidimetric and other photometric systems detect the initial aggregation of platelets by fibrinogen-platelet receptor GPIIb / IIIa binding, this may not reflect the final fibrin-platelet binding by GPIIb / IIIa receptor.

[0015] A significant of the limitations of systems using "normal" fibrinogen coated beads is that "normal" fibrinogen may not reflect both the amount and functionality of a particular patient's own fibrinogen. Therefore, fibrinogen-platelet receptor GPIIb / IIIa blocking measured by such systems is, however, an approximate estimation of fibrinogen-platelet GPIIb / IIIa blocking of the initial phase of platelet aggregation of an individual patient.

[0016] This is a significant limitation in some subsets of high-risk patients who may require treatment with an agent that inhibits platelet aggregation. they may have higher or lower levels of fibrinogen and therefore would need a careful assessment of platelet GPIIb / IIIa receptor blocking platelets to reduce bleeding complications caused by too low a rating of platelet GPIIb / IIIa receptor blocking or as a result of ischemic events too high a rating of platelet GPIIb / IIIa receptor blocking. In addition, fibrinogen levels and functionality may change during trauma associated with intervention procedures. At this time, accurate assessment of platelet GP IIb / III receptor blocking in real time, during and after the procedure, is required.

[0017] Thus, there is a need to develop a method and apparatus for measuring the effectiveness of antiplatelet agents continuously and throughout the hemostasis process from initial clot formation to lysis.

Brief description of the drawings [0018]

FIG. 1 is a graphic illustration depicting the mechanism of platelet aggregation.

FIG. 2 is a schematic diagram of a hemostasis analyzer in accordance with a preferred embodiment of the invention.

FIG. 3 is a graph illustrating the haemostatic profile generated by the haemostatic analyzer shown in Fig. 2.

FIGS. 4-10 are schematic illustrations depicting platelet activation in response to various antagonistic agents.

FIG. 11 illustrates several hemostatic profiles associated with platelet activation described in connection with Figs. 4-10

FIG. 12 is a schematic diagram of a hemostasis analyzer according to an alternative embodiment of the invention.

Detailed description of preferred embodiments [0019] The scope of protection is limited only by the claims.

[0020] According to preferred embodiments of the invention, a hemostasis analyzer, such as a Thrombelastograph® (TEG®) hemostasis analyzer available from Haemoscope Corp., Skokie, Illinois, is used to continuously measure the hemostasis process in real time, from initial fibrin formation by platelet-fibrin binding GPIIb / IIIa and lysis. Since several specific anti-platelet agents are discussed herein in connection with preferred embodiments of the invention, it should be understood that the invention is applicable in combination with virtually any anti-platelet agent. Furthermore, it will also be understood that the invention is applicable to measuring the effectiveness of coagulation or platelet activating agents.

[0021] According to preferred embodiments of the invention, the use of a hemostasis analyzer in accordance with the protocol of the invention allows: confirming the achievement of a therapeutic level of GPIIb / IIIa receptor blockade; individualizing the dosage estimation to assess the respective GPIIb / IIIa receptor blockade; individualizing the dose estimation is necessary to achieve adequate GPIIb / IIIa receptor blockade; depicting the rate of decrease in platelet inhibition or regeneration after inhibition after treatment with platelet inhibiting drugs; assessment of the effect of the combination of thrombolytic and platelet inhibiting agents in hemostasis in a patient.

[0022] The invention may use a hemostasis analyzer 10, such as the Thrombelastograph® (TEG®) hemostasis analyzer mentioned above, to measure the physical properties of the clot. An exemplary hemostasis analyzer 10 is described in detail in the above-mentioned US Patent 6,225,126 and is not fully discussed here. However, with reference to FIG. 2, to assist in understanding the invention, a brief description of the hemostasis analyzer 10 is provided. The haemostasis analyzer uses a special stationary cylindrical cup 12 in which blood samples 13 are stored. The cup 12 is connected to a drive mechanism that causes the cup to oscillate within an angle of 0, preferably around 4 ° 45 '. Each rotation cycle lasts 10 seconds. By means of the torsion tube 15, the needle 14 is suspended in the blood sample 13 and the movement of the needle 14 is monitored. The torque of the rotating cup 12 is transmitted to the immersed needle 14 only when the fibrin plate attachment binds the cup 12 and the needle 14 together. The strength of these fibrin-platelet bonds increases the movement of the needle, so that strong clots move the needle 14 directly in the cup movement phase. Therefore, the signal strength is directly related to the strength of the clot formed. When the clot shrinks or licks, these bonds break and the transfer of motion to the cup is reduced.

[0023] The rotation of the needle 14 is converted by the converter 16 into an electrical signal that can be monitored by a computer (not shown in FIG. 2) including the processor and the control program.

[0024] The computer may work on an electrical signal to create a hemostasis profile corresponding to the measured clotting process. In addition, the computer may include a display or may be connected to a printer to provide a visual representation of the haemostasis profile. This computer configuration is within the skill of one of ordinary skill in the art.

[0025] As will also be described, based on the assessment of the hemostasis profile, the computer through its control program may be adapted to provide dosage recommendations. As shown in FIG. 3, the resulting haemostasis profile 20 is a measurement of the time needed to form the first fibrin fiber, clot formation kinetics, clot strength (measured in millimeters (mm) and is converted into units of lateral elasticity dyn / cm²) and clot breakdown. Table I below provides the definition of several of these measured parameters.

Table I

<td>R</td><td>Time R is the latency time from the time the blood was placed in the TEG® analyzer to the initial formation of fibrin.</td>

<td>α</td><td>measure of fibrin accumulation and cross-linking (strengthening of the clot)</td>

<td>MA</td><td>MA, or maximum amplitude in mm, is a direct function of the maximum dynamic binding properties of GPIIb / IIIa fibrin and platelets and means the final strength of the fibrin clot.</td>

<td>LY30</td><td>LY30 measures the rate of amplitude reduction 30 minutes after MA and represents clot breakdown or lysis.</td>

[0026] Clinically, these measurements provide a vehicle for monitoring anticoagulant therapy (e.g. heparin or warfarin), thrombolytic therapy (e.g. tPA, streptokinase, urokinase), anti-fibrinolytic activity (e.g. ε-amino-caproic acid (Amicar®), trasylol (aprotinin ), tranexamic acid (TX)), antiplatelet agents (e.g. abciximab (ReoPro®), eptifibatide (INTEGRILIN®), tirofiban (Aggrastat®), blood component transfusion therapy, risk assessment of blood clotting in cancer and infection, high risk surgery and other conditions that may lead to excessive clotting (hypercoagulability) or excessive bleeding (low clotting conditions). Then, according to the invention, the hemostasis analyzer 10 is useful for testing the effectiveness of clinical drug therapy in stopping fibrinolysis, or the effectiveness of thrombolytic drugs for monitoring thrombolysis, the effectiveness of antiplatelet agents for monitoring platelet inhibition, ischemic or bleeding complications.

[0027] Hemostasis analyzer 10 and the associated computer plot of clot strength versus time at which the onset of clot formation, the reaction time (R) is quantitatively recorded (Fig. 3). This graph also shows the maximum clot strength (or stiffness) and MA of the blood sample. MA is a general estimation of platelet binding fibrifibrin GPIIb / IIIa, which is used, for example, for postoperative platelet or fibrinogen replacement therapy. Between platelets and fibrin alone, an abnormally low MA means that there are abnormalities in the platelets (i.e., quantitative or functional defects) and / or abnormalities in the blood fibrinogen content. However, by keeping fibrinogen and platelet levels constant, all MA changes will reflect changes in platelet function. Therefore, testing the same blood sample by two methods, one with antiplatelet agent and one without, will show the differences between the two MAs which will reflect the effect of the antiplatelet agent on platelet function.

[0028] Plaques play a key role in mediating ischemic complications resulting in stroke and myocardial infarction. Inhibition of platelet function by antiplatelet agents (platelet blocking drugs) such as aspirin, c7E3 Fab antibody fragment, abciximab (ReoPro®) or clopidogrel (Plavix®) can dramatically reduce the risk of death, myocardial infarction, or percutaneous endovascular reocclusion. coronary angioplasty (PTCA) or arterial thrombolytic therapy (IATT). Administration of excessive amounts of antiplatelet agents can lead to life-threatening hemorrhage. Therefore, an accurate estimation of a patient's inhibition of platelet function is very important for monitoring drug therapy because of the narrow risk / therapeutic ratio in this class of drugs.

[0029] By using the above strategy, which maintains fibrinogen and platelet counts constant, it is possible to properly administer and monitor antiplatelet drugs or to modify their doses or to measure fibrin contribution to MA (MAFIB) and by subtracting measurement of pure platelet content in MA ( MAp) as MAp = MA - MAfib. [0030] Therefore, according to preferred embodiments of the invention, to properly monitor antiplatelet agents, the following procedure should be followed:

1. The hemostasis analyzer, as it is commonly used, measures the function of platelets (MA or MAp), which are stimulated by thrombin, a strong platelet activator directly activating the GPIIb / IIIa receptor site. In order to sensitize MA or MAP to low platelet inhibition, platelet function should be activated by a less potent than thrombin platelet activator such as ADP, which indirectly activates the GPIIb / Ilia receptor site. Accordingly, when testing blood samples in a haemostasis analyzer in this case, thrombin formation is inhibited, for example, sodium citrate, heparin, hirudin, etc., and ADP is used to activate platelet function.

2. Unfortunately, thrombin is also involved in activating the conversion of fibrinogen to fibrin. Having suppressed thrombin in stage 1, it is necessary to use another enzyme to activate fibrinogen. A suitable enzyme is reptylase, whose only function is to activate the conversion of fibrinogen to fibrin. The clot is now stimulated by reptylase (activation of fibrinogen) and ADP (activation of platelets). The clot strength is measured by MA, and the contribution of the platelet function to the clot strength is measured as MAp, as described above.

3. The clot that is formed by a fibrinogen activator such as reptylase and a platelet activator such as ADP is usually weaker than that produced by thrombin. Therefore, you can choose the torsion cable 15 described above, which is more sensitive to a weaker clot and is able to measure changes in MA and MAp for the slight effect of antiplatelet agents such as ReoPro®. Alternatively, activated Factor XIII (Factor XIIIa) can be added. Factor XIIIa modifies the fibrin-platelet bond from a hydrogen bond to a stronger covalent bond, thereby increasing clot strength.

[0031] Based on the above, the following protocol can be implemented:

1. Necessary modification of the torsion wire in the hemostasis analyzer 10: production of wires with different torsion strength for different sensitivities in relation to shear force, suitable for measuring the effect of antiplatelet agents of different strength that can be measured. The sensitivity of the torsion wire is essentially related to its cross-section. To increase the sensitivity, the torsion wires have a cross-section that provides clot detection sensitivity in the range of about 150 to about 1000 dynes / cm² they are suitable for adaptation to the hemostasis analyzer described in the above-mentioned US Patent 6,225,236.

2. Reptylase-stimulated agonist-activated blood sample: Batroxabin (reptylase, Pentapharm) (15 μΐ prepared reptylase reagent) will be used and pre-added to cup 12 to activate fibrinogen to fibrin. In addition to Batroxabin, ADP (final concentration 20 μΜ) and 10 μΜ Factor XIIIa will be pre-added to cup 12. 340 μΐ whole blood with inhibited thrombin (e.g. citrate, heparinized, etc.) will be added to the heated cup 12 containing Batroxabin ADP and Factor XIIIa and the maximum clot strength will be estimated. In addition, a control sample that completely inhibits the effect of platelets on clot strength (MAfib) will also be exposed to Batroxabin and antiplatelet agent added to cup 12, providing a measure of the effect of fibrin on clot strength clotting strength in the absence of tensile stretching blood.

[0032] MApb was measured before treatment of the patient with the antiplatelet agent and MApa was measured after treatment. Platelet inhibition for drug effects will be calculated as follows:

MApb ~ MAb - MAfib

MApa = MAa - 'MAfib

Drug Inhibition = MApb + MAfib [0033] It will be understood by one of ordinary skill in the art that a torsion cord may be required for the measurement of clot strength described above, which is usually less sensitive under thrombin inhibition conditions. However, various test protocols may test for clots with a strength equal to or greater than typical thrombin-based. In such cases, the torsion cord 15 will be selected to be sensitive to such more durable clots. So torsion wires with several cross-sections can be used, providing a wide range of sensitivity from 100 dyne / cm2 to 25,000 dyne / cm2.

[0034] It should further be noted that the invention is applicable to measuring other clot formation parameters. For example, the haemostasis analyzer 10 measures the blood clotting process from the time the test was initiated, from the initial formation of fibrin, through the rate of clot strengthening and clot lysis. Therefore, according to the invention, it is possible to measure the effect of the presence of heparin by assessing the parameter R, which, as described above, shows inhibition of fibrin pre-formation. It is also possible to measure the effectiveness of drug therapy on thrombolytic activity by observing the LY30 parameter, which shows the clot lysis rate.

[0035] The existence of significant individual variability in the number of GPIIb / IIIa receptors per platelet and its ligand binding function is well documented. In addition, variable inhibition of GPIIb / IIIa function, due in part to differences in platelet counts, may occur after weight-adjusted platelet blocker dose administration. Subgroups of higher risk patients, such as diabetic patients undergoing percutaneous coronary angioplasty (PTCA), may require higher platelet blocker doses than are currently achieved after weight-matched blocker treatment, which is not individualized at this time to ensure adequate blocking GPIIb / IIIa receptor. Potential hemorrhagic or ischemic events indicate the need to individualize the estimation and development of the required dose to ensure that the therapeutic level of receptor blockade is achieved in real time. The apparatus and method according to preferred embodiments of the invention provides this possibility. [0036] In contrast to agents directed directly at inhibition of platelet GPIIb / IIIa receptor, Plavix® (clopidogrel) is a platelet antagonist of the adenosine diphosphate (ADP) receptor that inhibits the class of ADP receptors mediating platelet GPIIb / IIIa receptor activation. Plavix® is taken orally, usually as a loading dose of four 75 mg tablets, followed by long-term treatment with one 75 mg tablet daily before and after PTCA or patients at high risk of ischemic events. The Plavix® algorithm dictates the same dosage regardless of patient weight or hemostatic profile. Therefore, treatment with Plavix® may result in increased bleeding or failure to achieve an adequate therapeutic level of platelet inhibition. Accordingly, there is a need to prescribe and monitor individualized dosages of agents that inhibit both the GPIIb / IIIa platelet receptor and the ADP receptor (PI).

[0037] Another platelet agonist is Thromboxane A2 (TxA2), which activates the thromboxane A2 receptor. When the thromboxane A2 receptors are activated, they mediate the activation of GPIIb / IIIa receptors. Cyclooxygenase is an enzyme necessary for the production of thromboxane A2 and is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs). [0038] The result of the activated clotting protein is fibrin fiber, which together with activated platelets on GPIIb / IIIa forms a platelet fibrin (fibrinogen) binding to form the final clot. Therefore, in order for platelet fibrin (fibrinogen) to occur or to occur, GFIIb / IIIa receptors must be activated. Therefore, platelet agonists are constructed for GFIIb / IIIa receptor activation directly, through thrombin, or indirectly through ADP and thromboxane A2. In this regard, platelet inhibitory drugs are specifically directed to inhibiting these agonists as illustrated in FIG. 4-10.

[0039] With reference to FIG. 4-10, platelets 30 have a binding site on GPIIb / IIIa 32 receptor, ADP has a receptor binding site 34, and TxA2 has a receptor binding site 36. As shown in FIG. 4, the addition of an ADP receptor agonist activates the ADP receptor 34 binding site (shown by arrow 38), which activates the GPIIb / IIIa receptor binding site (shown by arrow 40). Platelet adenosine diphosphate (ADP) receptor agonist, such as Plavix®, ADP 34 receptor binding site (phantom arrow 42 in FIG. 5) and thus the GPIIb / IIIa site is not activated in response to the presence of an ADP agonist (phantom arrow 44) . Therefore, in the presence of an ADP receptor antagonist, the ADP agonist only activates platelets that are not inhibited. This reduces the clot strength, shown as underlined MApi in FIG. 11, compared to clot strength with full platelet activation shown as underlined MAkh in FIG. 11.

[0040] ReoPro®, Integrilin® and Aggrastat® inhibit the GPIIb / IIIa 32 receptor directly. When an ADP agonist is added, it activates the ADP receptor 34 (arrow 46 in FIG. 6) but stops at the GPIIb / IIIa receptor (phantom arrow 48). Therefore, in the presence of GPIIb / IIIa inhibitors, only uninhibited platelets will be activated by the ADP agonist causing correspondingly reduced clot strength, e.g., MApi shown in FIG. 11.

[0041] Thromboxane A2 activates the TxA2 platelet receptor 36 (arrow 50 in FIG. 7), which in turn activates the GPIIb / IIIa 32 receptor (arrow 52). Arachidonic acid (AA) is a precursor of thromboxane A2 and is converted to thromboxane A2 in the presence of cyclooxygenase. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (phantom arrow 54 in FIG. 8) and therefore in the presence of the TxA2 agonist (phantom arrow 56, the GPIIb / IIIa site is also not activated, resulting in a corresponding reduction in clot strength, e.g., MApi in FIG. 11. Therefore, AA platelet agonist can only activate the GPIIb / IIIa site when not NSAIDs 32 were adopted.

[0042] Thrombin is an enzyme that cleaves soluble fibrinogen into fibrin fibers. It is also the strongest platelet activator, strongly and directly increasing the expression and activation of platelet GPIIb / IIIa receptors (arrow 58 in FIG. 9). ReoPro®, Integrilin® and Aggrastat® inhibit the GPIIb / IIIa receptor response to ADP or TxA2 agonists; however, thrombin will still activate GPIIb / IIIa (arrow 60 in FIG. 10). Certain hemostasis assays, such as the TEG® assay described above, may be adapted to produce thrombin in the clot formation process. Increased expression and strong activation of platelet GPIIb / IIIa by thrombin overcomes GPIIb / IIIa inhibitors to provide clot strength at full platelet activation (MAkh in FIG. 11).

[0043] Based on the above discussion, it is therefore possible to accurately determine the protocol for monitoring platelet inhibitors such as GPIIb / IIIa platelet inhibitors, ADP and thromboxane A2. Referring to FIG. 12, apparatus 10 'may include a plurality of apparatus for hemostasis analysis, such as the hemostasis analyzer 10 shown in FIG. 2 and four are shown as devices 10a, 10b, 10c and 10d analyzing the respective blood samples 13a, 13b, 13c and 13d, respectively.

[0044] A first sample 13a of heparinized whole blood was prepared and loaded into the first hemostasis analyzer 10a. A fibrin 17 activator, such as a combination of reptylase and factor XIIIa, was added to sample cup 12. Activator 17 can be previously added to sample cup 12 or cup 12 can be treated with activator 17. Alternatively, activator 17 can be added to blood sample 13a after it is added to cup 12. Approximately 10 μΐ of activator 17 is added to 340μ1 sample blood. The test is completed and the strength of the resulting clot is measured. In the absence of any platelet agonist, this clot strength can be termed MAf, since it is only a contribution of fibrin with essentially no platelet activation.

[0045] A second sample 13b of heparinized whole blood was prepared and loaded into the second hemostasis analyzer 10b. It should be borne in mind that a homeostasis analyzer with a single chamber may be used in sequence for each test, an analyzer with more than one chamber may be used, or a multi-chamber analyzer may be used. For example, the TEG® haemostasis analyzer in standard configuration has two test chambers that can operate simultaneously. Two TEG® haemostasis analyzers may be used to perform each of four tests in accordance with this exemplary protocol, or one TEG® hemostasis analyzer may be used. What's more, two TEG® haemostasis analyzers can be networked to provide one device for testing four cells.

[0046] For the second sample 13b, the activator 17 is added to the sample, and, in a suitable proportion, the ADP agonist 18 is added to the second sample 13b. For example, 20μΜ of ADP agonist can be added to 340μ1 of the second heparinized whole blood sample. The test is completed and the strength of the resulting clot is measured. This clot strength can be described as MApi1 because it represents the contribution of fibrin and platelets not inhibited by the administration of any platelet-inhibiting ADP.

Due to the lack of any platelet agonist, this clot strength can be referred to as MAf, since it is only a contribution of fibrin without essentially platelet activation.

[0047] A third sample 13c of heparinized whole blood was prepared and loaded into the third 10c hemostasis anabzator. Fibrin 17 activator was added to sample 13c and also, in an appropriate proportion, a thromboxane A2 19 agonist was added to the third sample 13c. For example, 10 μ1 arachidonic acid (AA) can be added to 340 μ1 of a third heparinized whole blood sample. The test is completed and the strength of the resulting clot is measured. This clot strength can be termed MApi2 because it represents the contribution of fibrin and platelets not inhibited by the administration of any platelet-inhibiting TxA2 agent.

[0048] A fourth sample 13d of heparinized whole blood was prepared and loaded into the fourth 10d hemostasis analyzer. For the fourth sample 13d, heparinase 21 and kaolin 22 were used to neutralize the effect of heparin in the fourth sample 13d. The test is completed and the strength of the resulting clot is measured. This clot strength can be defined as MAkh, and it measures the maximum MA of platelet activation due to the use of heparinase and kaolin to neutralize heparin in the 13d sample which allows the production of thrombin which activates GPIIb / IIIa receptors.

[0049] The MApi-MAf value measures the unique contribution of PI inhibited platelets, where platelet inhibition occurs by Reopro®, Aggrastat®, Integrilin®, ADP and NSAIDs. The percentage reduction in MA due to platelet inhibition is then calculated for each of the MApi1 and MApi2 according to the equation:

Percentage of platelet activation = [(MApi - MAf) (MAkh - MAf)] * 100 where the MAkh-MAf value measures the unique share of fully activated platelets. In this way, the doctor can observe the effect of platelet inhibition therapy, isolated from the component effects and recommend appropriate dosages. Alternatively, the percentage of platelet inhibition can be set to 100 - [(MApi / -MAf) / MAkh-MAf)] * 100.

[0050] It will be understood that the sample cups 12 in the above tests can be prepared in advance and contain the appropriate means as described above in the protocol. In this regard, sample cups can be color coded to identify individual substances inside the cup. Sample cup sets 12 can be packaged to facilitate testing. In addition, media materials, e.g., activator 17, can be distributed in colored vials to facilitate identification and loading into sample cups 12, either before or after loading the appropriate blood sample into the sample cup.

[0051] The invention has been described with reference to several preferred embodiments. One skilled in the art will understand that the invention may be implemented in another manner without departing from its true scope which is set out in the appended claims.