Mechanisms of Thrombosis in Heparin-Induced Thrombocytopenia and Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombotic thrombocytopenia (VITT) are rare, iatrogenic immune-mediated conditions with high rates of thrombosis-related morbidity and mortality. HIT is a long-recognized reaction to the administration of the common parenterally administered anticoagulant heparin (or its derivatives), while VITT is a new, distinct syndrome occurring in response to adenovirus-based vaccines against coronavirus disease 2019 and potentially other types of vaccines. A feature of both HIT and VITT is paradoxical thrombosis despite a characteristic low platelet count, mediated by the presence of platelet-activating antibodies to platelet factor 4. Several additional factors have also been suggested to contribute to clot formation in HIT and/or VITT, including monocytes, tissue factor, microparticles, endothelium, the formation of neutrophil extracellular traps, complement, procoagulant platelets, and vaccine components. In this review, we discuss the literature to date regarding mechanisms contributing to thrombosis in both HIT and VITT and explore the pathophysiological similarities and differences between the two conditions.

Measurement of procoagulant platelets provides mechanistic insight and diagnostic potential in heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a prothrombotic, immune-mediated adverse drug reaction associated with high rates of thrombosis-related morbidity and mortality caused by FcγRIIa-activating pathogenic antibodies to PF4-heparin. Procoagulant platelets are a platelet subset that promote thrombin generation, are clinically relevant in prothrombotic diseases, and are formed when platelet G-protein-coupled receptor (GPCR) and ITAM-linked receptors are co-stimulated. OBJECTIVES: We examined the procoagulant platelet response of healthy donors to platelet agonists in the presence of HIT plasma and determined the contribution of FcγRIIa. PATIENTS/METHODS: Our previously established flow cytometry-based procoagulant platelet assay was modified to incorporate plasma samples, performed using FcγRIIa-responsive donor platelets. Plasma samples were serotonin-release assay-confirmed HIT (HIT+), or negative on HIT screening. RESULTS: In response to GPCR stimulation, only HIT+ plasma produced a heparin-dependent sensitization that required active FcγRIIa. As a potential diagnostic tool, the procoagulant platelet assay achieved 98% accuracy in identifying clinically verified HIT when performed blinded to the diagnoses of a validation cohort. Samples inducing a higher procoagulant platelet response were more likely from patients with thrombotic complications. Thrombin stimulation markedly increased the procoagulant platelet response with HIT+ plasma that was heparin independent and only partially reversed by FcγRIIa blockade, possibly reflecting ongoing thrombotic risk after heparin cessation. CONCLUSIONS: We demonstrate that HIT plasma together with platelet agonists increased the procoagulant platelet proportions, which may contribute to thrombotic risk in HIT. Targeting procoagulant platelet activation may represent a novel treatment strategy. This assay may be a rapid, clinically relevant functional assay for accurately detecting pathological HIT antibodies.

The PAR4 Platelet Thrombin Receptor Variant rs773902 does not Impact the Incidence of Thrombotic or Bleeding Events in a Healthy Older Population.

BACKGROUND: Protease-activated receptor 4 (PAR4) is a platelet thrombin receptor important for thrombosis and a target of antiplatelet drug development. A frequently occurring single-nucleotide polymorphism (rs773902) causes a PAR4 sequence variant (NC_000019.10:p.Ala120Thr) whereby platelets from Thr120-expressing individuals are hyperresponsive to PAR4 agonists versus platelets from Ala120-expressing individuals. However, whether this enhanced platelet responsiveness translates to increased thrombotic risk or decreased bleeding risk remains unknown. OBJECTIVES: This article examines the association of rs773902 with adjudicated cardiovascular events and aspirin use in a randomized trial population of healthy older individuals. METHODS: We analyzed 13,547 participants in the ASPirin in Reducing Events in the Elderly trial. Participants had no previous cardiovascular events at enrollment and were randomized to either 100 mg daily aspirin or placebo for a median follow-up of 4.7 years. Total genotypes were 8,761 (65%) GG (Ala120 variant), 4,303 (32%) heterozygotes, and 483 (4%) AA (Thr120 variant). Cox proportional hazard regression tested the relationship between rs773902 and thrombotic events (major adverse cardiovascular events [MACE] and ischemic stroke [IS]) and bleeding (major hemorrhage [MHEM] and intracranial bleeding [ICB]). RESULTS: No statistically significant association was observed overall or by treatment group between rs773902 and any thrombotic or bleeding event examined. Further, there was no significant interaction between rs773902 and treatment for any of MACE, IS, MHEM, or ICB. CONCLUSION: This post hoc analysis of a prospective cohort study suggests that, despite sensitizing platelet activation, the rs773902 PAR4 variant is not associated with thrombotic cardiovascular or bleeding events in a healthy older population.

Disrupting the platelet internal membrane via PI3KC2α inhibition impairs thrombosis independently of canonical platelet activation.

Arterial thrombosis causes heart attacks and most strokes and is the most common cause of death in the world. Platelets are the cells that form arterial thrombi, and antiplatelet drugs are the mainstay of heart attack and stroke prevention. Yet, current drugs have limited efficacy, preventing fewer than 25% of lethal cardiovascular events without clinically relevant effects on bleeding. The key limitation on the ability of all current drugs to impair thrombosis without causing bleeding is that they block global platelet activation, thereby indiscriminately preventing platelet function in hemostasis and thrombosis. Here, we identify an approach with the potential to overcome this limitation by preventing platelet function independently of canonical platelet activation and in a manner that appears specifically relevant in the setting of thrombosis. Genetic or pharmacological targeting of the class II phosphoinositide 3-kinase (PI3KC2α) dilates the internal membrane reserve of platelets but does not affect activation-dependent platelet function in standard tests. Despite this, inhibition of PI3KC2α is potently antithrombotic in human blood ex vivo and mice in vivo and does not affect hemostasis. Mechanistic studies reveal this antithrombotic effect to be the result of impaired platelet adhesion driven by pronounced hemodynamic shear stress gradients. These findings demonstrate an important role for PI3KC2α in regulating platelet structure and function via a membrane-dependent mechanism and suggest that drugs targeting the platelet internal membrane may be a suitable approach for antithrombotic therapies with an improved therapeutic window.

The PI 3-kinase PI3KC2α regulates mouse platelet membrane structure and function independently of membrane lipid composition.

PI3KC2α is a phosphoinositide 3-kinase with a recently reported function in platelets; PI3KC2α-deficient mouse platelets have altered membrane structure and impaired function. Yet, how these membrane changes cause platelet dysfunction remains unknown. Here, focused ion beam-scanning electron microscopy of PI3KC2α-deficient platelet ultrastructure reveals a specific effect on the internal membrane structure, while liquid chromatography-tandem mass spectrometry profiling of 294 lipid species shows unaltered lipid composition. Functionally, PI3KC2α-deficient platelets exhibit impaired thrombosis specifically under conditions involving membrane tethering. These studies indicate that the structural changes in PI3KC2α-deficient platelets are limited to the membrane, occur without major changes in lipid composition, and selectively impair cell function during thrombus formation. These findings illustrate a unique mechanism that may be targetable for anti-thrombotic benefit.

Structure and function of the open canalicular system - the platelet's specialized internal membrane network.

The open canalicular system (OCS) is an internal membrane structure found in platelets. First identified 50 years ago, the OCS comprises a tunneling network of surface-connected channels that appear to play an important role in platelet function. Yet, our understanding of how the OCS forms, how it functions, and what might regulate its structure and behavior remains fairly rudimentary. Structural abnormalities of the OCS are observed in some human platelet disorders. Yet, because platelets from these patients display multiple defects, the specific contribution of any OCS dysregulation to the impaired platelet function is unclear. However, recent studies have begun to shed light on mechanisms that regulate the OCS structure and to understand what influence the OCS has on overall platelet function. Advances in cellular imaging techniques have allowed whole-cell visualization of the OCS, providing the opportunity for a more detailed structural examination. Furthermore, recent work indicates that the modulation of the OCS structure may be sufficient to impact in vivo platelet function, opening up the intriguing possibility of manipulating the OCS structure as an anti-thrombotic approach. On the 50th anniversary of its discovery, we review here what is known about OCS structure and function, and outline some of the key microscopy tools for studying this intriguing internal membrane system.