Intravital Assessment of Blood Platelet Function. A Review of the Methodological Approaches with Examples of Studies of Selected Aspects of Blood Platelet Function.

Platelet biology owes to intravital studies not only a better understanding of platelets' role in primary hemostasis but also findings that platelets are important factors in inflammation and atherosclerosis. Researchers who enter the field of intravital platelet studies may be confused by the heterogeneity of experimental protocols utilized. On the one hand, there are a variety of stimuli used to activate platelet response, and on the other hand there are several approaches to measure the outcome of the activation. A number of possible combinations of activation factors with measurement approaches result in the aforementioned heterogeneity. The aim of this review is to present the most often used protocols in a systematic way depending on the stimulus used to activate platelets. By providing examples of studies performed with each of the protocols, we attempt to explain why a particular combination of stimuli and measurement method was applied to study a given aspect of platelet biology.

Predictive value of platelet reactivity unit (PRU) value for thrombotic and hemorrhagic events during flow diversion procedures: a meta-analysis.

BACKGROUND AND PURPOSE: Platelet function testing prior to flow diversion procedures, although initially heavily debated, has seen a substantial increase in its adoption to assess the risk of operative and perioperative thrombotic and hemorrhagic events. This meta-analysis was conducted to assess platelet function testing, particularly the VerifyNow Platelet Reactivity Unit (PRU) assay, for a relationship between the reported assay PRU value and thrombotic and hemorrhagic events. MATERIALS AND METHODS: The currently available literature (2013-2018) was surveyed with PubMed and Google Scholar searches. Included studies were those for which there were at least 30 cases during the study period, for which VerifyNow platelet reactivity unit values were obtained prior to the procedures and for which intraoperative and perioperative adverse events were noted. PRU value cut-offs ranging from >200 to >240 comprised the hyporesponse group while values ranging from <60 to <70 comprised the hyper-response group. The data were subject to statistical analysis to assess the relationship between PRU values and thrombotic and hemorrhagic events. The collected data were subsequently statistically analyzed to assess for publication bias. RESULTS: The searches yielded 27 studies, of which 12 met the inclusion criteria for the meta-analysis. The meta-analysis included data from 1464 reported Pipeline cases. The study included 273 men and 1177 women with a mean age across the analyzed procedures of 58 years (range 25-85). After loading with antiplatelet medications, preprocedural platelet hyper-responsiveness was associated with a greater incidence of hemorrhagic events with an increased absolute risk of 12%, but showed no relationship with thrombotic events. Preprocedural platelet hyporesponsiveness was associated with a greater incidence of thrombotic events with an absolute risk of 15%, but showed no relationship with hemorrhagic events. CONCLUSIONS: VerifyNow PRU values that correspond to platelet hyporesponse or hyper-response to dual antiplatelet therapy are associated with a higher risk of thrombotic and hemorrhagic events, respectively. Thus, the PRU value may offer some predictive value for these events.

Platelet Function Tests.

Traditionally developed for diagnosis of bleeding disorders, platelet function assays have become increasingly used in basic research on platelet physiology, in phenotype-genotype associations in bleeding disorders, in drug development as surrogate endpoints of efficacy of new antiplatelet therapy, and to an extent, in the monitoring of antiplatelet therapy in clinical practice to predict thrombotic and bleeding risk. A multiplicity of platelet function assays is available to measure the level of platelet activity in various settings. These include assays that are restricted to a specialized laboratory as well as point-of-care instruments meant to investigate platelet function at patient bedside. Unlike tests that determine a defined quantity or measurement of a clinical biomarker (e.g., cholesterol or blood pressure), platelet function testing assesses the dynamics of living cells, which immediately presents a series of unique problems to any laboratory or clinic. This article presents currently used platelet function assays and discusses important variables to take into account when performing these assays, including preanalytical issues and difficulties in interpreting platelet function test results.

Platelet function tests: a review of progresses in clinical application.

The major goal of traditional platelet function tests has been to screen and diagnose patients who present with bleeding problems. However, as the central role of platelets implicated in the etiology of arterial thrombotic diseases such as myocardial infarction and stroke became widely known, platelet function tests are now being promoted to monitor the efficacy of antiplatelet drugs and also to potentially identify patients at increased risk of thrombosis. Beyond hemostasis and thrombosis, an increasing number of studies indicate that platelets play an integral role in intercellular communication, are mediators of inflammation, and have immunomodulatory activity. As new potential biomarkers and technologies arrive at the horizon, platelet functions testing appears to take on a new aspect. This review article discusses currently available clinical application of platelet function tests, placing emphasis on essential characteristics.

Efficacy and safety of intensified antiplatelet therapy on the basis of platelet reactivity testing in patients after percutaneous coronary intervention: systematic review and meta-analysis.

BACKGROUND: ADP-specific platelet function assays were shown to predict thrombotic events, and might be helpful to select candidates for more potent antiplatelet therapy. We aimed to determine the efficacy and safety of giving intensified antiplatelet therapy on the basis of platelet reactivity testing for patients undergoing percutaneous coronary intervention (PCI). METHODS: Electronic databases were searched to find prospective, randomized trials that reported the clinical impact of using an intensified antiplatelet protocol (repeated loading or elevated maintenance doses of clopidogrel, prasugrel or glycoprotein IIb/IIIa inhibitor) on the basis of ADP-specific platelet reactivity testing (VerifyNow, Multiplate, VASP or light transmission aggregometry) compared to standard-dose clopidogrel. Evaluated efficacy measures included cardiovascular death, non-fatal myocardial infarction and definite/probable stent thrombosis (ST), while major bleeding events were recorded as safety endpoint. RESULTS: Between 2008 and 2011, 10 clinical trials comprising 4213 randomized patients were identified. Compared to standard antiplatelet therapy, the intensified protocol was associated with a significant reduction in cardiovascular mortality, ST and myocardial infarction (p<0.01 for all). There was no difference in the rate of major bleeding events between intensified and standard groups (p=0.44). Although the observed effects regarding mortality, ST and bleeding were not heterogeneous, meta-regression analysis revealed that the net clinical benefit of the intensified treatment significantly depended on the risk of ST with standard-dose clopidogrel (p=0.023). CONCLUSION: Intensifying antiplatelet therapy on the basis of platelet reactivity testing reduces cardiovascular mortality and ST after PCI; however, the net benefit of this approach depends on the risk of ST with standard-dose clopidogrel.

Role of platelet function testing in clinical practice: current concepts and future perspectives.

Oral antiplatelet therapy is routinely administered to ACS patients as well as to patients undergoing percutaneous coronary intervention (PCI) with the primary aim of inhibiting platelet-mediated thrombus formation and subsequent abrupt vessel occlusion. Individual platelet response to aspirin and especially to clopidogrel is highly variable and evidence has grown in recent years linking an attenuated response to therapy with the occurrence of ischemic events. At present, the antiplatelet therapy landscape is changing with the emergence of prasugrel and ticagrelor as alternative and more potent treatment options. In addition, tests for near-patient monitoring of platelet function in clinical practice are available and are being increasingly employed for the optimization of antiplatelet treatment. It is hypothesized that platelet function testing may prove useful for achieving an optimized balance of proven platelet inhibition at a cost of moderate bleeding risk. This is also why first centers have already included testing in day-to-day routine. Extensive clinical evaluations with a range of currently-available assays for platelet function testing are ongoing and the current and future role of platelet function testing in clinical practice is a topic of much debate. Widespread adoption of this practice and its incorporation into clinical guidelines awaits the results of ongoing trials where treatment is changed based on platelet function testing data. This review paper summarizes the key characteristics of platelet function tests available, presents an overview of relevant studies and examines the present role of platelet function testing in clinical practice with a focus on antiplatelet therapy in patients undergoing coronary stent placement.

New trends in bioanalytical microdevices to assess platelet function.

Cardiovascular disease is the major cause of mortality globally. The role of platelets and antiplatelet drugs in the treatment of cardiovascular disease is widely appreciated. Platelets have a less well-known role in cancer and inflammation and as the role of platelets in cancer and inflammation is increasingly understood, there is a compelling need to develop diagnostic assays of platelet function to guide clinical management. Most current platelet-function tests are focused on platelet aggregation, are cumbersome, require dedicated laboratory personnel, measure a single response to a single agonist and artificially separate platelets from blood, thus, these assays do not reflect the pathophysiolgical environment of complex disease states. New technology platforms are emerging that address the physiological adhesive function of platelets to vascular-specific matrices using small volumes of whole blood, giving rapid results. These technologies will guide therapy in the prevention of cardiovascular disease and probably in risk management in both cancer and atherosclerosis.

Physiology of hemostasis: with relevance to current and future laboratory testing.

This article briefly details the physiologic and interdependent mechanisms of vascular hemostasis, with an eye toward how the laboratory can assist in diagnosing and maintaining the balance of procoagulant and anticoagulant functions. These functions include determining characteristics of the blood vessel wall, platelet components and receptor-ligand interactions critical for hemostasis, the regulation of thrombin generation and its effects, and the complex fibrinolytic pathways that complete the coagulation cascade.

Clopidogrel resistance: a diagnostic challenge.

No clear and generally accepted definition of clopidogrel resistance has been achieved up to now. We believe that the new point-of-care assays of platelet function may help the clinicians to overcome to define clopidogrel resistance especially in patients undergoing percutaneous coronary intervention in whom the level of inhibition of platelets is clinically essential.

New approaches in the measurement of coagulation.

In this session contributors present recent developments in laboratory tools for investigation of haemorrhagic disorders as well as their relative utility in clinical research. In an overview of B. Sørensen the present knowledge is summarized on the dynamic properties of whole blood fibrin formation as studied by changes in whole blood elasticity on a thrombelastometry system. Additionally, fibrin formation dynamics using simple APTT methods are presented. G. Castaman reviews the pathophysiology of von Willebrand's disease (VWD) and explains which tests are best used in diagnosis and subclassification of VWD accounting for recent developments. This presentation also describes the treatment technologies available today and their implications in clinical management of bleeding episodes in VWD. J. Lloyd addresses the assay discrepancy phenomenon that is found in some of our patients suffering from mild haemophilia A. Assay discrepancy most often means a much lower factor VIII:C value by a two-stage or chromogenic assay for factor VIII:C compared to the activity recorded by the one-stage clotting system for factor VIII:C. In rare cases, the opposite phenomenon exist. The presentation includes data from 16 Australian families with discrepant results. D. Varon reports on an assay for study of platelet function in whole blood under flow conditions. The equipment is described as a cone-and-plate(let) analyser in which the adhesion and aggregation of platelets onto a polystyrene surface is studied under arterial flow conditions. Basically, it is anticipated that proteins such as VWF and fibrinogen of flowing blood is attached to the polystyrene surface where they build up a thrombogenic surface. In the study of the author platelets were pre-activated with agonists and platelet deposits were determined after passage of whole blood for a pre-set time interval. Data presented suggest that the assay is sensitive to platelet numbers as well as qualitative changes in platelets themselves, and several examples of disorders characterized by enhanced as well as reduced platelet aggregating activities illustrates the sensitivity of the method.

Tests of platelet function.

PURPOSE OF REVIEW: Platelets play a vital role in the normal hemostasis, and derangements of their function can lead to hemorrhage or thrombosis. While we have made progress in elucidating the molecular mechanisms leading to platelet adhesion, aggregation, shape change, and secretion, clinically useful tests of platelet function have lagged behind. The following is a review of some of the currently available tests of platelet function, their advantages and drawbacks, as well as the clinical scenarios in which they are likely to be useful. RECENT FINDINGS: Attention is now being paid to standardization and optimization of older tests such as platelet aggregometry, in addition to better defining the role of newer tests such as the platelet function analyzer and thromboelastography in diagnosing and managing disorders of primary hemostasis and platelet function. SUMMARY: Platelet function is complex and may be disrupted at any of a number of steps, including adhesion, aggregation, shape change and secretion. We are better defining the role of the currently available tests, while identifying gaps in our ability to diagnose disorders of platelet function.

[Research advance in platelet function assays].

Platelets play a key role in the pathogenesis of atherothrombosis, and also perform the physiological hemostasis. The platelet function assays have values in the investigating patients with suspected platelet disorders and in studying the effects of anti-platelet drugs. There are increasing assays for investigating platelet function, including assessment of platelet adhesion, activation and aggregation, etc. However, all of these assays have certain limited sensitivity. It is necessary to develop a simple, sensitive assay that measures the activated platelet. This article reviewed advances of researches on platelet function assays, including assay for general function of platelet, assay of platelet adhesion function, platelet activation assay, platelet aggregation assay, platelet coagulation assay and application of flow cytometry in assessment of platelet functions, etc. and looked forward to research prospect in this field.

The platelet function analyzer (PFA-100): an update on its clinical use.

The platelet function analyzer, PFA-100, is a relatively new method which has been developed as a quantitative, simple and rapid in vitro tool of assessing primary hemostasis. The aim of this review is to summarize the published studies reporting on the utility of the PFA-100 device as a screening tool for primary hemostasis. Data were identified by searches of the published literature, including PubMed, references from reviews and abstracts from the most important meetings on this topic. The literature data document that the PFA-100 is a useful screening tool for the investigation of von Willebrand's disease and various acquired and congenital intrinsic platelet function disorders. It is also useful for evaluating primary hemostasis before surgical procedures and for monitoring desmopressin therapy in patients with type 1 von Willebrand's disease. Moreover, recent studies have shown its potential for therapeutic monitoring of the effectiveness of anti-platelet medications in cardiovascular disease management. Given its high sensitivity, speed and simplicity of use, we conclude that the PFA-100 could replace the in vivo bleeding time as a screening test for primary hemostasis in routine clinical practice.